Optimal soybean loci

ABSTRACT

As disclosed herein, optimal native genomic loci have been identified in dicot plants, such as soybean plants, that represent best sites for targeted insertion of exogenous sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit, under 35 U.S.C. § 119(e), to U.S.Provisional Patent Application No. 61/899,566, filed on Nov. 4, 2013 andU.S. Provisional Patent Application No. 61/889,587, filed on Nov. 4,2013, the contents of which are incorporated by reference in theirentirety into the present application.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“232306seqlist.txt”, created on Oct. 31, 2014, and having a size of 13.7megabytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

REFERENCE TO TABLE LISTING SUBMITTED ELECTRONICALLY

The official copy of the table listing is submitted electronically viaEFS-Web as a .PDF formatted table listing with a file named “Table 3”,created on Nov. 4, 2013, and having a size of 12 megabytes and is filedconcurrently with the specification. The table listing contained in this.PDF formatted document is part of the specification and is hereinincorporated by reference in its entirety.

BACKGROUND

The genome of numerous types of dicot plants, for example soybeanplants, was successfully transformed with transgenes in the early1990's. Over the last twenty years, numerous methodologies have beendeveloped for transforming the genome of dicot plants, like soybean,wherein a transgene is stably integrated into the genome of dicotplants. This evolution of dicot transformation methodologies hasresulted in the capability to successfully introduce a transgenecomprising an agronomic trait within the genome of dicot plants, such assoybean. The introduction of insect resistance and herbicide toleranttraits within dicot plants in the late-1990's provided producers with anew and convenient technological innovation for controlling insects anda wide spectrum of weeds, which was unparalleled in cultivation farmingmethods. Currently, transgenic dicot plants are commercially availablethroughout the world, and new transgenic products such as Enlist™Soybean offer improved solutions for ever-increasing weed challenges.The utilization of transgenic dicot plants in modern agronomic practiceswould not be possible, but for the development and improvement oftransformation methodologies.

However, current transformation methodologies rely upon the randominsertion of transgenes within the genome of dicot plants, such assoybean. Reliance on random insertion of genes into a genome has severaldisadvantages. The transgenic events may randomly integrate within genetranscriptional sequences, thereby interrupting the expression ofendogenous traits and altering the growth and development of the plant.In addition, the transgenic events may indiscriminately integrate intolocations of the genome that are susceptible to gene silencing,culminating in the reduced or complete inhibition of transgeneexpression either in the first or subsequent generations of transgenicplants. Finally, the random integration of transgenes within the plantgenome requires considerable effort and cost in identifying the locationof the transgenic event and selecting transgenic events that perform asdesigned without agronomic impact to the plant. Novel assays must becontinually developed to determine the precise location of theintegrated transgene for each transgenic event, such as a soybeantransgenic event. The random nature of plant transformationmethodologies results in a “position-effect” of the integratedtransgene, which hinders the effectiveness and efficiency oftransformation methodologies.

Targeted genome modification of plants has been a long-standing andelusive goal of both applied and basic research. Targeting genes andgene stacks to specific locations in the genome of diot plants, such assoybean plants, will improve the quality of transgenic events, reducecosts associated with production of transgenic events and provide newmethods for making transgenic plant products such as sequential genestacking. Overall, targeting trangenes to specific genomic sites islikely to be commercially beneficial. Significant advances have beenmade in the last few years towards development of methods andcompositions to target and cleave genomic DNA by site specific nucleases(e.g., Zinc Finger Nucleases (ZFNs), Meganucleases, TranscriptionActivator-Like Effector Nucelases (TALENS) and Clustered RegularlyInterspaced Short Palindromic Repeats/CRISPR-associated nuclease(CRISPR/Cas) with an engineered crRNA/tracr RNA), to induce targetedmutagenesis, induce targeted deletions of cellular DNA sequences, andfacilitate targeted recombination of an exogenous donor DNApolynucleotide within a predetermined genomic locus. See, for example,U.S. Patent Publication No. 20030232410; 20050208489; 20050026157;20050064474; and 20060188987, and International Patent Publication No.WO 2007/014275, the disclosures of which are incorporated by referencein their entireties for all purposes. U.S. Patent Publication No.20080182332 describes use of non-canonical zinc finger nucleases (ZFNs)for targeted modification of plant genomes and U.S. Patent PublicationNo. 20090205083 describes ZFN-mediated targeted modification of a plantEPSPs genomic locus. Current methods for targeted insertion of exogenousDNA typically involve co-transformation of plant tissue with a donor DNApolynucleotide containing at least one transgene and a site specificnuclease (e.g., ZFN) which is designed to bind and cleave a specificgenomic locus of an actively transcribed coding sequence. This causesthe donor DNA polynucleotide to stably insert within the cleaved genomiclocus resulting in targeted gene addition at a specified genomic locuscomprising an actively transcribed coding sequence.

An alternative approach is to target the transgene to preselected targetnongenic loci within the genome of dicot plants like soybean. In recentyears, several technologies have been developed and applied to plantcells for the targeted delivery of a transgene within the genome ofdicot plants like soybean. However, much less is known about theattributes of genomic sites that are suitable for targeting.Historically, non-essential genes and pathogen (viral) integration sitesin genomes have been used as loci for targeting. The number of suchsites in genomes is rather limiting and there is therefore a need foridentification and characterization of targetable optimal genomic locithat can be used for targeting of donor polynucleotide sequences. Inaddition to being amenable to targeting, optimal genomic loci areexpected to be neutral sites that can support transgene expression andbreeding applications. A need exists for compositions and methods thatdefine criteria to identify optimal nongenic loci within the genome ofdicot plants, for example soybean plants, for targeted transgeneintegration.

SUMMARY

One embodiment of the present disclosure is directed to methods ofidentifying optimal sites in a dicot genome, including for example thesoybean genome for the insertion of exogenous sequences. There isdocumentation in the literature that suggests that plant chromosomalregions are targetable and support expression. Applicants haveconstructed a set of criteria for identifying regions of native soybeangenomic sequences that are optimal sites for site directed targetedinsertion. More particularly, in accordance with one embodiment, anoptimal locus should be nongenic, targetable, support gene expression,agronomically neutral, and have evidence of recombination. As disclosedherein, applicants have discovered a number of loci in the soybeangenome that meet these criteria and thus represent optimal sites for theinsertion of exogenous sequences.

In accordance with one embodiment a recombinant soybean sequence isdisclosed herein wherein the recombinant sequence comprises a nongenicsoybean genomic sequence of at least 1 Kb, and a DNA of interestinserted into the nongenic soybean genomic sequence, wherein thenongenic soybean genomic sequence has been modified by the insertion ofthe DNA of interest. In one embodiment the native nongenic soybeansequence is hypomethylated, expressable, exemplifies evidence ofrecombination and is located in proximal location to a genic region inthe soybean genome. In one embodiment the nongenic sequence has a lengthranging from about 1 Kb to about 5.7 Kb. In one embodiment the DNA ofinterest comprises exogenous DNA sequences, including for exampleregulatory sequences, restriction cleavage sites, RNA encoding regionsor protein encoding regions. In one embodiment the DNA of interestcomprises a gene expression cassette comprising one or more transgenes

In accordance with one embodiment a recombinant sequence is providedcomprising an optimal nongenic soybean genomic sequence of about 1 Kb toabout 5.7 Kb and a DNA of interest wherein the nongenic soybean genomicsequence has 1, 2, 3, 4 or 5 of the following properties orcharacteristics:

a) has a known or predicted soybean coding sequence within 40 Kb of saidsoybean genomic sequence;

b) has a sequence comprising a 2 Kb upstream and/or 1 Kb downstream of aknown soybean gene within 40 Kb of one end of said soybean genomicsequence;

c) does not contain greater than 1% DNA methylation within the sequence;d) does not contain a 1 Kb sequence having greater than 40% sequenceidentity to any other sequence within the soybean genome; and

e) exemplifies evidence of recombination at a recombination frequency ofgreater than 0.01574 cM/Mb.

In accordance with one embodiment a soybean plant, soybean plant part,or soybean plant cell is provided, comprising a DNA of interest insertedinto an identified and targeted nongenic soybean genomic sequence of thesoybean plant, soybean plant part, or soybean plant cell. In oneembodiment the nongenic soybean genomic sequence of the soybean plant,soybean plant part, or soybean plant cell is hypomethylated,expressable, exemplifies evidence of recombination and is located inproximal location to an genic region in the soybean genome. In oneembodiment the nongenic soybean genomic sequence of the soybean plant,soybean plant part, or soybean plant cell is about 1 Kb to about 5.7 Kbin length, is hypomethylated and has 1, 2, 3 or 4 of the followingproperties or characteristics:

a) has a known or predicted soybean coding sequence within 40 Kb of saidsoybean genomic sequence;

b) has a sequence comprising a 2 Kb upstream and/or 1 Kb downstream of aknown soybean gene within 40 Kb of one end of said soybean genomicsequence;

c) does not contain greater than 1% DNA methylation within the sequence;

d) does not comprise a 1 Kb sequence having greater than 40% sequenceidentity to any other sequence within the soybean genome; and

e) exemplifies evidence of recombination at a recombination frequency ofgreater than 0.01574 cM/Mb.

In one embodiment a method of making a transgenic plant cell comprisinga DNA of interest targeted to a nongenic soybean genomic sequence isprovided, the method comprising:

a) selecting an optimal nongenic soybean genomic locus;

b) introducing a site specific nuclease into a plant cell, wherein thesite specific nuclease cleaves said nongenic sequence;

c) introducing the DNA of interest into the plant cell;

d) targeting the DNA of interest into said nongenic sequence, whereinthe cleavage of said nongenic sequence stimulates integration of thepolynucleotide sequence into said nongenic sequence; and

e) selecting transgenic plant cells comprising the DNA of interesttargeted to said nongenic sequence.

In accordance with one embodiment the selected nongenic sequencecomprises 2, 3, 4, 5, 6, 7 or 8 of the following characteristics:

a) the nongenic sequence does not contain a methylated polynucleotide;

b) the nongenic sequence exhibits a 0.01574 to 83.52 cM/Mb rate ofrecombination within the soybean genome;

c) the nongenic sequence exhibits a 0 to 0.494 level of nucleosomeoccupancy of the soybean genome;

d) the nongenic sequence shares less than 40% sequence identity with anyother 1 Kb sequence contained in the soybean genome;

e) the nongenic sequence has a relative location value from 0 to 0.99682ratio of genomic distance from a soybean chromosomal centromere;

f) the nongenic sequence has a guanine/cytosine percent content range of14.36 to 45.9%;

g) the nongenic sequence is located proximally to a genic sequence; and,

h) a 1 Mb region of soybean genomic sequence comprising said nongenicsequence, comprises one or more nongenic sequences.

An embodiment of the present disclosure is directed to methods ofidentifying a nongenic soybean genomic sequence, comprising the stepsof:

a) identifying soybean genomic sequences of at least 1 Kb in length thatdo not contain greater than a 1% level of methylation to generate afirst pool of sequences;

b) eliminating any soybean genomic sequences that encode soybeantranscripts from the first pool of sequences;

c) eliminating any soybean genomic sequences that do not provideevidence of recombination from the first pool of sequences;

d) eliminating any soybean genomic sequences that comprise a 1 Kbsequence that shares 40% or higher sequence identity with another 1 Kbsequence contained in the soybean genome from the first pool ofsequences;

e) eliminating any soybean genomic sequence that do not have a knownsoybean gene within 40 Kb of the identified sequence from the first poolof sequences; and,

f) identifying the remaining soybean genomic sequences in the pool ofsequences as nongenic soybean genomic sequence. Once the sequences havebeen identified they can be manipulated using recombinant techniques totarget the insertion of nucleic acid sequences not found in the loci inthe native genome.

In accordance with an embodiment, any soybean genomic sequences that donot have a known soybean gene, or at least a 2 Kb upstream or 1 Kbdownstream sequence of a known gene located within 40 Kb of the soybeangenomic sequence are eliminated from the pool of nongenic soybeangenomic sequences.

In accordance with an embodiment, any soybean genomic sequences that donot have a gene expressing a soybean protein located within 40 Kb of thesoybean genomic sequence are eliminated from the pool of nongenicsoybean genomic sequences.

In accordance with one embodiment a purified soybean polynucleotidesequence is disclosed herein wherein the purified sequence comprises anongenic soybean genomic sequence of at least 1 Kb. In one embodimentthe nongenic soybean sequence is hypomethylated, expressable,exemplifies evidence of recombination and is located in proximallocation to a genic region in the soybean genome. In one embodiment thenongenic sequence has a length ranging from about 1 Kb to about 5.7 Kb.In one embodiment the DNA of interest comprises exogenous DNA sequences,including for example regulatory sequences, restriction cleavage sites,RNA encoding regions or protein encoding regions. In one embodiment theDNA of interest comprises a gene expression cassette comprising one ormore transgenes.

In accordance with one embodiment a purified soybean polynucleotidesequence is provided comprising an optimal nongenic soybean genomicsequence of about 1 Kb to about 5.7 Kb and a DNA of interest wherein thenongenic soybean genomic sequence has 1, 2, 3, 4 or 5 of the followingproperties or characteristics:

a) has a known or predicted soybean coding sequence within 40 Kb of saidrecombinant sequence;

b) has a sequence comprising a 2 Kb upstream and/or 1 Kb downstream of aknown soybean gene within 40 Kb of one end of said nongenic;

c) does not contain a methylated polynucleotide;

d) does not contain a 1 Kb sequence having greater than 40% sequenceidentity to any other sequence within the soybean genome; and

e) exemplifies evidence of recombination at a recombination frequency ofgreater than 0.01574 cM/Mb.

In accordance with one embodiment, a purified soybean polynucleotidesequence is provided comprising a selected nongenic sequence. Theselected nongenic sequence comprises 2, 3, 4, 5, 6, 7 or 8 of thefollowing characteristics:

a) the nongenic sequence does not contain a methylated polynucleotide;

b) the nongenic sequence exhibits a 0.01574 to 83.52 cM/Mb rate ofrecombination within the soybean genome;

c) the nongenic sequence exhibits a 0 to 0.494 level of nucleosomeoccupancy of the soybean genome;

d) the nongenic sequence shares less than 40% sequence identity with anyother 1 Kb sequence contained in the soybean genome;

e) the nongenic sequence has a relative location value from 0 to 0.99682ratio of genomic distance from a soybean chromosomal centromere;

the nongenic sequence has a guanine/cytosine percent content range of14.36 to 45.9%;

g) the nongenic sequence is located proximally to a genic sequence; and,

h) a 1 Mb region of soybean genomic sequence comprising said nongenicsequence, comprises one or more nongenic sequences.

In accordance with an embodiment, any soybean genomic sequences that donot provide evidence of recombination at a recombination frequency ofgreater than 0.01574 cM/Mb are eliminated from the pool of nongenicsoybean genomic sequences.

In accordance with one embodiment the selected nongenic sequencecomprise the following characteristics:

a) the nongenic sequence does not contain greater than 1% DNAmethylation within the sequence

b) the nongenic sequence has a relative location value from 0.211 to0.976 ratio of genomic distance from a soybean chromosomal centromere;

c) the nongenic sequence has a guanine/cytosine percent content range of25.62 to 43.76%; and,

d) the nongenic sequence is from about 1 Kb to about 4.4 Kb in length.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Represents a three dimensional graph of the 7,018 select genomicloci clustered into 32 clusters. The clusters can be graphed threedimensionally and distinguished by color or other indicators. Eachcluster was assigned a unique identifier for ease of visualization,wherein all select genomic loci with the same identifier belonging tothe same cluster. After the clustering process, a representative selectgenomic loci was chosen from each cluster. This was performed bychoosing a select genomic loci, within each cluster, that was closest tothe centroid of that cluster.

FIG. 2. Provides a schematic drawing indicating the chromosomaldistribution of the optimal genomic loci, selected for being closest tothe centroid of each of the 32 respective clusters.

FIG. 3. Provides a schematic drawing indicating the soybean chromosomallocation of the optimal genomic loci selected for targeting validation.

FIG. 4. Representation of the universal donor polynucleotide sequencefor integration via non-homologous end joining (NHEJ). Two proposedvectors are provide wherein a DNA of interest (DNA X) comprises one ormore (i.e., “1-N”) zinc finger binding sites (ZFN BS) at either end ofthe DNA of interest. Vertical arrows show unique restriction sites andhorizontal arrows represent potential PCR primer sites.

FIG. 5. Representation of the universal donor polynucleotide sequencefor integration via homologous-directed repair (HDR). A DNA of interest(DNA X) comprising two regions of homologous sequences (HA) flanking theDNA of interest with zinc finger nuclease binding sites (ZFN) bracketingthe DNAX and HA sequences. Vertical arrows show unique restriction sitesand horizontal arrows represent potential PCR primer sites.

FIG. 6. Validation of soybean selected genomic loci targets using NHEJbased Rapid Targeting Analysis (RTA) method.

FIG. 7. Plasmid map of pDAB124280 (SEQ ID NO:7561). The numberedelements (i.e., GmPPL01ZF391R and GMPPL01ZF391L) correspond with zincfinger nuclease binding sequences of about 20 to 35 base pairs in lengththat are recognized and cleaved by corresponding zinc finger nucleaseproteins. These zinc finger binding sequences and the annotated “UZISequence” (which is a 100-150 bp template region containing restrictionsites and DNA sequences for primer design or coding sequences) comprisethe universal donor cassette. Further included in this plasmid design isthe “104113 Overlap” which are sequences that share homology to theplasmid vector for high throughput assembly of the universal donorcassettes within a plasmid vector (i.e., via Gibson assembly).

FIG. 8. Plasmid map of pDAB124281 (SEQ ID NO:7562). The numberedelements (i.e., GmPPL02ZF411R and GMPPL02ZF411L) correspond with zincfinger nuclease binding sequences of about 20 to 35 base pairs in lengththat are recognized and cleaved by corresponding zinc finger nucleaseproteins. These zinc finger binding sequences and the annotated “UZISequence” (which is a 100-150 bp template region containing restrictionsites and DNA sequences for primer design or coding sequences) comprisethe universal donor cassette. Further included in this plasmid design isthe “104113 Overlap” which are sequences that share homology to theplasmid vector for high throughput assembly of the universal donorcassettes within a plasmid vector (i.e., via Gibson assembly).

FIG. 9. Plasmid map of pDAB121278 (SEQ ID NO:7563). The numberedelements (i.e., GmPPL18_4 and GMPPL18_3) correspond with zinc fingernuclease binding sequences of about 20 to 35 base pairs in length thatare recognized and cleaved by corresponding zinc finger nucleaseproteins. These zinc finger binding sequences and the annotated “UZISequence” (which is a 100-150 bp template region containing restrictionsites and DNA sequences for primer design or coding sequences) comprisethe universal donor cassette. Further included in this plasmid design isthe “104113 Overlap” which are sequences that share homology to theplasmid vector for high throughput assembly of the universal donorcassettes within a plasmid vector (i.e., via Gibson assembly).

FIG. 10. Plasmid map of pDAB123812 (SEQ ID NO:7564). The numberedelements (i.e., ZF538R and ZF538L) correspond with zinc finger nucleasebinding sequences of about 20 to 35 base pairs in length that arerecognized and cleaved by corresponding zinc finger nuclease proteins.These zinc finger binding sequences and the annotated “UZI Sequence”(which is a 100-150 bp template region containing restriction sites andDNA sequences for primer design or coding sequences) comprise theuniversal donor cassette. Further included in this plasmid design is the“104113 Overlap” which are sequences that share homology to the plasmidvector for high throughput assembly of the universal donor cassetteswithin a plasmid vector (i.e., via Gibson assembly).

FIG. 11. Plasmid map of pDAB121937 (SEQ ID NO:7565). The numberedelements (i.e., GmPPL34ZF598L, GmPPL34ZF598R, GmPPL36ZF599L,GmPPL36ZF599R, GmPPL36ZF600L, and GmPPL36ZF600R) correspond with zincfinger nuclease binding sequences of about 20 to 35 base pairs in lengththat are recognized and cleaved by corresponding zinc finger nucleaseproteins. These zinc finger binding sequences and the annotated “UZISequence” (which is a 100-150 bp template region containing restrictionsites and DNA sequences for primer design or coding sequences) comprisethe universal donor cassette. Further included in this plasmid design isthe “104113 Overlap” which are sequences that share homology to theplasmid vector for high throughput assembly of the universal donorcassettes within a plasmid vector (i.e., via Gibson assembly).

FIG. 12. Plasmid map of pDAB123811 (SEQ ID NO:7566). The numberedelements (i.e., ZF 560L and ZF 560R) correspond with zinc fingernuclease binding sequences of about 20 to 35 base pairs in length thatare recognized and cleaved by corresponding zinc finger nucleaseproteins. These zinc finger binding sequences and the annotated “UZISequence” (which is a 100-150 bp template region containing restrictionsites and DNA sequences for primer design or coding sequences) comprisethe universal donor cassette. Further included in this plasmid design isthe “104113 Overlap” which are sequences that share homology to theplasmid vector for high throughput assembly of the universal donorcassettes within a plasmid vector (i.e., via Gibson assembly).

FIG. 13. Plasmid map of pDAB124864 (SEQ ID NO:7567). The numberedelements (i.e., ZF631L and ZF631R) correspond with zinc finger nucleasebinding sequences of about 20 to 35 base pairs in length that arerecognized and cleaved by corresponding zinc finger nuclease proteins.These zinc finger binding sequences and the annotated “UZI Sequence”(which is a 100-150 bp template region containing restriction sites andDNA sequences for primer design or coding sequences) comprise theuniversal donor cassette. Further included in this plasmid design is the“104113 Overlap” which are sequences that share homology to the plasmidvector for high throughput assembly of the universal donor cassetteswithin a plasmid vector (i.e., via Gibson assembly).

FIG. 14. Plasmid map of pDAB7221 (SEQ ID NO:7569). This plasmid containsthe Cassava Vein Mosaic Virus Promoter (CsVMV) driving the GFP proteinand flanked by the Agrobacterium tumefaciens (AtuORF 24 3′UTR).

FIGS. 15A-15C. Histrogram of characteristics (length, expression ofcoding region within 40 Kb of loci, and recombination frequency) for theidentified optimal nongenic soybean loci. FIG. 15A illustrates adistribution of the polynucleotide sequence lengths of the optimalgenomic loci (OGL). FIG. 15B illustrates the distribution of the optimalnongenic maize loci relative to their recombination frequency. FIG. 15Cillustrates the distribution of expressed nucleic acid sequencesrelative to their proximity (log scale) to the optimal genomic loci(OGL).

DETAILED DESCRIPTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

As used herein, the term “plant” includes a whole plant and anydescendant, cell, tissue, or part of a plant. The term “plant parts”include any part(s) of a plant, including, for example and withoutlimitation: seed (including mature seed and immature seed); a plantcutting; a plant cell; a plant cell culture; a plant organ (e.g.,pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, andexplants). A plant tissue or plant organ may be a seed, callus, or anyother group of plant cells that is organized into a structural orfunctional unit. A plant cell or tissue culture may be capable ofregenerating a plant having the physiological and morphologicalcharacteristics of the plant from which the cell or tissue was obtained,and of regenerating a plant having substantially the same genotype asthe plant. In contrast, some plant cells are not capable of beingregenerated to produce plants. Regenerable cells in a plant cell ortissue culture may be embryos, protoplasts, meristematic cells, callus,pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears,cobs, husks, or stalks.

Plant parts include harvestable parts and parts useful for propagationof progeny plants. Plant parts useful for propagation include, forexample and without limitation: seed; fruit; a cutting; a seedling; atuber; and a rootstock. A harvestable part of a plant may be any usefulpart of a plant, including, for example and without limitation: flower;pollen; seedling; tuber; leaf; stem; fruit; seed; and root.

A plant cell is the structural and physiological unit of the plant.Plant cells, as used herein, includes protoplasts and protoplasts with acell wall. A plant cell may be in the form of an isolated single cell,or an aggregate of cells (e.g., a friable callus and a cultured cell),and may be part of a higher organized unit (e.g., a plant tissue, plantorgan, and plant). Thus, a plant cell may be a protoplast, a gameteproducing cell, or a cell or collection of cells that can regenerateinto a whole plant. As such, a seed, which comprises multiple plantcells and is capable of regenerating into a whole plant, is considered a“plant part” in embodiments herein.

The term “protoplast”, as used herein, refers to a plant cell that hadits cell wall completely or partially removed, with the lipid bilayermembrane thereof naked. Typically, a protoplast is an isolated plantcell without cell walls which has the potency for regeneration into cellculture or a whole plant.

As used herein the terms “native” or “natural” define a condition foundin nature. A “native DNA sequence” is a DNA sequence present in naturethat was produced by natural means or traditional breeding techniquesbut not generated by genetic engineering (e.g., using molecularbiology/transformation techniques).

As used herein, “endogenous sequence” defines the native form of apolynucleotide, gene or polypeptide in its natural location in theorganism or in the genome of an organism.

The term “isolated” as used herein means having been removed from itsnatural environment.

The term “purified”, as used herein relates to the isolation of amolecule or compound in a form that is substantially free ofcontaminants normally associated with the molecule or compound in anative or natural environment and means having been increased in purityas a result of being separated from other components of the originalcomposition. The term “purified nucleic acid” is used herein to describea nucleic acid sequence which has been separated from other compoundsincluding, but not limited to polypeptides, lipids and carbohydrates.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

As used herein an “optimal dicot genomic loci”, “optimal nongenic dicotloci”, “optimal nongenic loci”, or “optimal genomic loci (OGL)” is anative DNA sequence found in the nuclear genome of a dicot plant thathas the following properties: nongenic, hypomethylated, targetable, andin proximal location to a genic region, wherein the genomic regionaround the optimal dicot genomic loci exemplifies evidence ofrecombination.

As used herein the terms “optimal soybean genomic loci”, “optimalnongenic soybean loci”, “optimal nongenic loci”, or “optimal genomicloci (OGL)” are used interchangably to designatea native DNA sequencefound in the nuclear genome of a dicot plant that has the followingproperties: nongenic, hypomethylated, targetable, and in proximallocation to a genic region, wherein the genomic region around theoptimal dicot genomic loci exemplifies evidence of recombination.

As used herein, the terms “nongenic dicot sequence” or “nongenic dicotgenomic sequence” are used interchangably to designatea native DNAsequence found in the nuclear genome of a dicot plant, having a lengthof at least 1 Kb, and devoid of any open reading frames, gene sequences,or gene regulatory sequences. Furthermore, the nongenic dicot sequencedoes not comprise any intron sequence (i.e., introns are excluded fromthe definition of nongenic). The nongenic sequence cannot be transcribedor translated into protein. Many plant genomes contain nongenic regions.As much as 95% of the genome can be nongenic, and these regions may becomprised of mainly repetitive DNA.

As used herein, the terms “nongenic soybean sequence” or “nongenicsoybean genomic sequence” are used interchangably to designatea nativeDNA sequence found in the nuclear genome of a soybean plant, having alength of at least 1 Kb, and devoid of any open reading frames, genesequences, or gene regulatory sequences. Furthermore, the nongenicsoybean sequence does not comprise any intron sequence (i.e., intronsare excluded from the definition of nongenic). The nongenic sequencecannot be transcribed or translated into protein. Many plant genomescontain nongenic regions. As much as 95% of the genome can be nongenic,and these regions may be comprised of mainly repetitive DNA.

As used herein, a “genic region” is defined as a polynucleotide sequencethat comprises an open reading frame encoding an RNA and/or polypeptide.The genic region may also encompass any identifiable adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation of expressionof the open reading frame up to about 2 Kb upstream of the coding regionand 1 Kb downstream of the coding region, but possibly further upstreamor downstream. A genic region further includes any introns that may bepresent in the genic region. Further, the genic region may comprise asingle gene sequence, or multiple gene sequences interspersed with shortspans (less than 1 Kb) of nongenic sequences.

As used herein a “nucleic acid of interest”, “DNA of interest”, or“donor” is defined as a nucleic acid/DNA sequence that has been selectedfor site directed, targeted insertion into the dicot genome, like asoybean genome. A nucleic acid of interest can be of any length, forexample between 2 and 50,000 nucleotides in length (or any integer valuetherebetween or thereabove), preferably between about 1,000 and 5,000nucleotides in length (or any integer value therebetween). A nucleicacid of interest may comprise one or more gene expression cassettes thatfurther comprise actively transcribed and/or translated gene sequences.Conversely, the nucleic acid of interest may comprise a polynucleotidesequence which does not comprise a functional gene expression cassetteor an entire gene (e.g., may simply comprise regulatory sequences suchas a promoter), or may not contain any identifiable gene expressionelements or any actively transcribed gene sequence. The nucleic acid ofinterest may optionally contain an analytical domain. Upon insertion ofthe nucleic acid of interest into the dicot genome of soybean forexample, the inserted sequences are referred to as the “inserted DNA ofinterest”. Further, the nucleic acid of interest can be DNA or RNA, canbe linear or circular, and can be single-stranded or double-stranded. Itcan be delivered to the cell as naked nucleic acid, as a complex withone or more delivery agents (e.g., liposomes, poloxamers, T-strandencapsulated with proteins, etc.,) or contained in a bacterial or viraldelivery vehicle, such as, for example, Agrobacterium tumefaciens or anadenovirus or an adeno-associated Virus (AAV), respectively.

As used herein the term “analytical domain” defines a nucleic acidsequence that contains functional elements that assist in the targetedinsertion of nucleic acid sequences. For example, an analytical domainmay contain specifically designed restriction enzyme sites, zinc fingerbinding sites, engineered landing pads or engineered transgeneintegration platforms and may or may not comprise gene regulatoryelements or an open reading frame. See, for example, U.S. PatentPublication No 20110191899, incorporated herein by reference in itsentirety.

As used herein the term “selected dicot sequence” defines a nativegenomic DNA sequence of a dicot plant that has been chosen for analysisto determine if the sequence qualifies as an optimal nongenic dicotgenomic loci.

As used herein the term “selected soybean sequence” defines a nativegenomic DNA sequence of a soybean plant that has been chosen foranalysis to determine if the sequence qualifies as an optimal nongenicsoybean genomic loci.

As used herein, the term “hypomethylation” or “hypomethylated”, inreference to a DNA sequence, defines a reduced state of methylated DNAnucleotide residues in a given sequence of DNA. Typically, the decreasedmethylation relates to the number of methylated adenine or cytosineresidues, relative to the average level of methylation found in nongenicsequences present in the genome of a dicot plant like a soybean plant.

As used herein a “targetable sequence” is a polynucleotide sequence thatis sufficiently unique in a nuclear genome to allow site specific,targeted insertion of a nucleic acid of interest into one specificsequence.

As used herein the term “non-repeating” sequence is defined as asequence of at least 1 Kb in length that shares less than 40% identityto any other sequence within the genome of a dicot plant, like soybean.Calculations of sequence identity can be determined using any standardtechnique known to those skilled in the art including, for example,scanning a selected genomic sequence against the dicot genome, e.g.,soybean c.v. Williams82 genome, using a BLAST™ based homology searchusing the NCBI BLAST™+ software (version 2.2.25) run using the defaultparameter settings (Stephen F. Altschul et al (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402). For example, as the selected soybeansequences (from the Glycine max c.v. Williams82 genome) were analyzed,the first BLAST™ hit identified from such a search represents the dicotsequence, e.g., soybean c.v. Williams82 sequence, itself. The secondBLAST™ hit for each selected soybean sequence was identified and thealignment coverage (represented as the percent of the selected soybeansequence covered by the BLAST™ hit) of the hit was used as a measure ofuniqueness of the selected soybean sequence within the genome of a dicotplant, such as soybean. These alignment coverage values for the secondBLAST™ hit ranged from a minimum of 0% to a maximum of 39.97% sequenceidentity. Any sequences that aligned at higher levels of sequenceidentity were not considered.

The term “in proximal location to a genic region” when used in referenceto a nongenic sequence defines the relative location of the nongenicsequence to a genic region. Specifically, the number of genic regionswithin a 40 Kb neighborhood (i.e., within 40 Kb on either end of theselected optimal soybean genomic loci sequence) is analyzed. Thisanalysis was completed by assaying gene annotation information and thelocations of known genes in the genome of a known dicot, such assoybean, that were extracted froma moncot genome database, for examplethe Soybean Genome Database. For each of the optimal nongenic soybeangenomic loci, e.g., 7,018 optimal nongenic soybean genomic loci, a 40 Kbwindow around the optimal genomic loci sequence was defined and thenumber of annotated genes with locations overlapping this window wascounted. The number of genic regions ranged from a minimum of 1 gene toa maximum of 18 genes within the 40 Kb neighborhood.

The term “known soybean coding sequence” as used herein relates to anypolynucleotide sequence identified from any dicot genomic database,including the Soybean Genomic Database (www.soybase.org, Shoemaker, R.C. et al. SoyBase, the USDA-ARS soybean genetics and genomics database.Nucleic Acids Res. 2010 January: 38 (Database issue):D843-6.) thatcomprise an open reading frame, either before or after processing ofintron sequences, and are transcribed into mRNA and optionallytranslated into a protein sequence when placed under the control of theappropriate genetic regulatory elements. The known soybean codingsequence can be a cDNA sequence or a genomic sequence. In someinstances, the known soybean coding sequence can be annotated as afunctional protein. In other instances, the known soybean codingsequence may not be annotated.

The term “predicted dicot coding sequence” as used herein relates to anyExpressed Sequence Tag (EST) polynucleotide sequences described in adicot genomic database, for example the Soybean Genomic Database. ESTsare identified from cDNA libraries constructed using oligo(dT) primersto direct first-strand synthesis by reverse transcriptase. The resultingESTs are single-pass sequencing reads of less than 500 bp obtained fromeither the 5′ or 3′ end of the cDNA insert. Multiple ESTs may be alignedinto a single contig. The identified EST sequences are uploaded into thedicot genomic database, e.g., Soybean Genomic Database and can besearched via bioinformatics methods to predict corresponding genomicpolynucleotide sequences that comprise a coding sequence that istranscribed into mRNA and optionally translated into a protein sequencewhen placed under the control of the appropriate genetic regulatoryelements.

The term “predicted soybean coding sequence” as used herein relates toany Expressed Sequence Tag (EST) polynucleotide sequences described in asoybean genomic database, for example the Soybean Genomic Database. ESTsare identified from cDNA libraries constructed using oligo(dT) primersto direct first-strand synthesis by reverse transcriptase. The resultingESTs are single-pass sequencing reads of less than 500 bp obtained fromeither the 5′ or 3′ end of the cDNA insert. Multiple ESTs may be alignedinto a single contig. The identified EST sequences are uploaded into thesoybean genomic database, e.g., Soybean Genomic Database and can besearched via bioinformatics methods to predict corresponding genomicpolynucleotide sequences that comprise a coding sequence that istranscribed into mRNA and optionally translated into a protein sequencewhen placed under the control of the appropriate genetic regulatoryelements.

The term “evidence of recombination” as used herein relates to themeiotic recombination frequencies between any pair of dicot genomicmarkers, e.g., soybean genomic markers, across a chromosome regioncomprising the selected soybean sequence. The recombination frequencieswere calculated based on the ratio of the genetic distance betweenmarkers (in centimorgan (cM)) to the physical distance between themarkers (in megabases (Mb)). For a selected soybean sequence to haveevidence of recombination, the selected soybean sequence must contain atleast one recombination event between two markers flanking the selectedsoybean sequence as detected using a high resolution marker datasetgenerated from multiple mapping populations.

As used herein the term “relative location value” is a calculated valuedefining the distance of a genomic locus from its correspondingchromosomal centromere. For each selected soybean sequence, the genomicdistance from the native location of the selected soybean sequence tothe centromere of the chromosome that it is located on, is measured (inBp). The relative location of selected soybean sequence within thechromosome is represented as the ratio of its genomic distance to thecentromere relative to the length of the specific chromosomal arm(measured in Bp) that it lies on. These relative location values for theoptimal nongenic soybean genomic loci can be generated for differentdicot plants, the relative location values for the soybean datasetranged from a minimum of 0 to a maximum of 0.99682 ratio of genomicdistance.

The term “exogenous DNA sequence” as used herein is any nucleic acidsequence that has been removed from its native location and insertedinto a new location altering the sequences that flank the nucleic acidsequence that has been moved. For example, an exogenous DNA sequence maycomprise a sequence from another species.

“Binding” refers to a sequence-specific, interaction betweenmacromolecules (e.g., between a protein and a nucleic acid). Not allcomponents of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (Kd). “Affinity”refers to the strength of binding: increased binding affinity beingcorrelated with a lower binding constant (Kd).

A “binding protein” is a protein that is able to bind to anothermolecule. A binding protein can bind to, for example, a DNA molecule (aDNA-binding protein), an RNA molecule (an RNA-binding protein) and/or aprotein molecule (a protein-binding protein). In the case of aprotein-binding protein, it can bind to itself (to form homodimers,homotrimers, etc.) and/or it can bind to one or more molecules of adifferent protein or proteins. A binding protein can have more than onetype of binding activity. For example, zinc finger proteins haveDNA-binding, RNA-binding and protein-binding activity.

As used herein the term “zinc fingers,” defines regions of amino acidsequence within a DNA binding protein binding domain whose structure isstabilized through coordination of a zinc ion.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP. Zinc finger bindingdomains can be “engineered” to bind to a predetermined nucleotidesequence. Non-limiting examples of methods for engineering zinc fingerproteins are design and selection. A designed zinc finger protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. See, for example, U.S. Pat. Nos. 6,140,081;6,453,242; 6,534,261 and 6,794,136; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. See, e.g.,U.S. Patent Publication No. 20110301073, incorporated by referenceherein in its entirety.

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system. Briefly, a “CRISPR DNAbinding domain” is a short stranded RNA molecule that acting in concerwith the CAS enzyme can selectively recognize, bind, and cleave genomicDNA. The CRISPR/Cas system can be engineered to create a double-strandedbreak (DSB) at a desired target in a genome, and repair of the DSB canbe influenced by the use of repair inhibitors to cause an increase inerror prone repair. See, e.g., Jinek et al (2012) Science 337, p.816-821, Jinek et al, (2013), eLife 2:e00471, and David Segal, (2013)eLife 2:e00563).

Zinc finger, CRISPR and TALE binding domains can be “engineered” to bindto a predetermined nucleotide sequence, for example via engineering(altering one or more amino acids) of the recognition helix region of anaturally occurring zinc finger. Similarly, TALEs can be “engineered” tobind to a predetermined nucleotide sequence, for example by engineeringof the amino acids involved in DNA binding (the repeat variablediresidue or RVD region). Therefore, engineered DNA binding proteins(zinc fingers or TALEs) are proteins that are non-naturally occurring.Non-limiting examples of methods for engineering DNA-binding proteinsare design and selection. A designed DNA binding protein is a proteinnot occurring in nature whose design/composition results principallyfrom rational criteria. Rational criteria for design include applicationof substitution rules and computerized algorithms for processinginformation in a database storing information of existing ZFP and/orTALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication Nos.20110301073, 20110239315 and 20119145940.

A “selected” zinc finger protein, CRISPR or TALE is a protein not foundin nature whose production results primarily from an empirical processsuch as phage display, interaction trap or hybrid selection. See e.g.,U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No.6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO01/60970 WO 01/88197 and WO 02/099084 and U.S. Publication Nos.20110301073, 20110239315 and 20119145940.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides, including but not limited to, donor captureby non-homologous end joining (NHEJ) and homologous recombination. Forthe purposes of this disclosure, “homologous recombination (HR)” refersto the specialized form of such exchange that takes place, for example,during repair of double-strand breaks in cells via homology-directedrepair mechanisms. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the nucleotide sequence that experienced the double-strand break), andis variously known as “non-crossover gene conversion” or “short tractgene conversion,” because it leads to the transfer of geneticinformation from the donor to the target. Without wishing to be bound byany particular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide. For HR-directed integration, the donor moleculecontains at least 2 regions of homology to the genome (“homology arms”)of least 50-100 base pairs in length. See, e.g., U.S. Patent PublicationNo. 20110281361.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break for HR mediated integration or having no homology to thenucleotide sequence in the region of the break for NHEJ mediatedintegration, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingerproteins, CRISPRS or TALEN can be used for additional double-strandedcleavage of additional target sites within the cell.

Any of the methods described herein can be used for insertion of a donorof any size and/or partial or complete inactivation of one or moretarget sequences in a cell by targeted integration of donor sequencethat disrupts expression of the gene(s) of interest. Cell lines withpartially or completely inactivated genes are also provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence (transgene) may produce one or more RNAmolecules (e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis),microRNAs (miRNAs), etc.) or protein.

“Cleavage” as used herein defines the breakage of the phosphate-sugarbackbone of a DNA molecule. Cleavage can be initiated by a variety ofmethods including, but not limited to, enzymatic or chemical hydrolysisof a phosphodiester bond. Both single-stranded cleavage anddouble-stranded cleavage are possible, and double-stranded cleavage canoccur as a result of two distinct single-stranded cleavage events. DNAcleavage can result in the production of either blunt ends or staggeredends. In certain embodiments, fusion polypeptides are used for targeteddouble-stranded DNA cleavage. A “cleavage domain” comprises one or morepolypeptide sequences which possesses catalytic activity for DNAcleavage. A cleavage domain can be contained in a single polypeptidechain or cleavage activity can result from the association of two (ormore) polypeptides.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528, 2008/0131962 and2011/0201055, incorporated herein by reference in their entireties.

A “target site” or “target sequence” refers to a portion of a nucleicacid to which a binding molecule will bind, provided sufficientconditions for binding exist.

Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

A “product of an exogenous nucleic acid” includes both polynucleotideand polypeptide products, for example, transcription products(polynucleotides such as RNA) and translation products (polypeptides).

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, for example, covalently. The subunit molecules can be thesame chemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid. Expression of a fusion protein in a cell can result from deliveryof the fusion protein to the cell or by delivery of a polynucleotideencoding the fusion protein to a cell, wherein the polynucleotide istranscribed, and the transcript is translated, to generate the fusionprotein. Trans-splicing, polypeptide cleavage and polypeptide ligationcan also be involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

For the purposes of the present disclosure, a “gene”, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent or operably linked to coding and/ortranscribed sequences. Accordingly, a gene includes, but is notnecessarily limited to, promoter sequences, terminators, translationalregulatory sequences such as ribosome binding sites and internalribosome entry sites, enhancers, silencers, insulators, boundaryelements, replication origins, matrix attachment sites and locus controlregions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, interfering RNA, ribozyme, structural RNA or any other type of RNA)or a protein produced by translation of a mRNA. Gene products alsoinclude RNAs which are modified, by processes such as capping,polyadenylation, methylation, and editing, and proteins modified by, forexample, methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

Sequence identity: The term “sequence identity” or “identity,” as usedherein in the context of two nucleic acid or polypeptide sequences,refers to the residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.

As used herein, the term “percentage of sequence identity” refers to thevalue determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences, and amino acid sequences) over a comparisonwindow, wherein the portion of the sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleotide oramino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described in, for example:Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch(1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad.Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higginsand Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res.16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearsonet al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMSMicrobiol. Lett. 174:247-50. A detailed consideration of sequencealignment methods and homology calculations can be found in, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-10. The National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST™; Altschul et al. (1990)) is available from several sources,including the National Center for Biotechnology Information (Bethesda,Md.), and on the internet, for use in connection with several sequenceanalysis programs. A description of how to determine sequence identityusing this program is available on the internet under the “help” sectionfor BLAST™. For comparisons of nucleic acid sequences, the “Blast 2sequences” function of the BLAST™ (Blastn) program may be employed usingthe default parameters. Nucleic acid sequences with even greatersimilarity to the reference sequences will show increasing percentageidentity when assessed by this method.

Specifically hybridizable/Specifically complementary: As used herein,the terms “specifically hybridizable” and “specifically complementary”are terms that indicate a sufficient degree of complementarity, suchthat stable and specific binding occurs between the nucleic acidmolecule and a target nucleic acid molecule. Hybridization between twonucleic acid molecules involves the formation of an anti-parallelalignment between the nucleic acid sequences of the two nucleic acidmolecules. The two molecules are then able to form hydrogen bonds withcorresponding bases on the opposite strand to form a duplex moleculethat, if it is sufficiently stable, is detectable using methods wellknown in the art. A nucleic acid molecule need not be 100% complementaryto its target sequence to be specifically hybridizable. However, theamount of sequence complementarity that must exist for hybridization tobe specific is a function of the hybridization conditions used.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na+ and/or Mg++ concentration) of thehybridization buffer will determine the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are known to those of ordinary skill in the art, and arediscussed, for example, in Sambrook et al. (ed.) Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames andHiggins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985.Further detailed instruction and guidance with regard to thehybridization of nucleic acids may be found, for example, in Tijssen,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” in Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, Part I,Chapter 2, Elsevier, N.Y., 1993; and Ausubel et al., Eds., CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, NY, 1995.

As used herein, “stringent conditions” encompass conditions under whichhybridization will only occur if there is less than 20% mismatch betweenthe hybridization molecule and a sequence within the target nucleic acidmolecule. “Stringent conditions” include further particular levels ofstringency. Thus, as used herein, “moderate stringency” conditions arethose under which molecules with more than 20% sequence mismatch willnot hybridize; conditions of “high stringency” are those under whichsequences with more than 10% mismatch will not hybridize; and conditionsof “very high stringency” are those under which sequences with more than5% mismatch will not hybridize. The following are representative,non-limiting hybridization conditions.

High Stringency condition (detects sequences that share at least 90%sequence identity): Hybridization in 5×SSC buffer (wherein the SSCbuffer contains a detergent such as SDS, and additional reagents likesalmon sperm DNA, EDTA, etc.) at 65° C. for 16 hours; wash twice in2×SSC buffer (wherein the SSC buffer contains a detergent such as SDS,and additional reagents like salmon sperm DNA, EDTA, etc.) at roomtemperature for 15 minutes each; and wash twice in 0.5×SSC buffer(wherein the SSC buffer contains a detergent such as SDS, and additionalreagents like salmon sperm DNA, EDTA, etc.) at 65° C. for 20 minuteseach.

Moderate Stringency condition (detects sequences that share at least 80%sequence identity): Hybridization in 5×-6×SSC buffer (wherein the SSCbuffer contains a detergent such as SDS, and additional reagents likesalmon sperm DNA, EDTA, etc.) at 65-70° C. for 16-20 hours; wash twicein 2×SSC buffer (wherein the SSC buffer contains a detergent such asSDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at roomtemperature for 5-20 minutes each; and wash twice in 1×SSC buffer(wherein the SSC buffer contains a detergent such as SDS, and additionalreagents like salmon sperm DNA, EDTA, etc.) at 55-70° C. for 30 minuteseach.

Non-stringent control condition (sequences that share at least 50%sequence identity will hybridize): Hybridization in 6×SSC buffer(wherein the SSC buffer contains a detergent such as SDS, and additionalreagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55°C. for 16-20 hours; wash at least twice in 2×-3×SSC buffer (wherein theSSC buffer contains a detergent such as SDS, and additional reagentslike salmon sperm DNA, EDTA, etc.) at room temperature to 55° C. for20-30 minutes each.

As used herein, the term “substantially homologous” or “substantialhomology,” with regard to a contiguous nucleic acid sequence, refers tocontiguous nucleotide sequences that hybridize under stringentconditions to the reference nucleic acid sequence. For example, nucleicacid sequences that are substantially homologous to a reference nucleicacid sequence are those nucleic acid sequences that hybridize understringent conditions (e.g., the Moderate Stringency conditions setforth, supra) to the reference nucleic acid sequence. Substantiallyhomologous sequences may have at least 80% sequence identity. Forexample, substantially homologous sequences may have from about 80% to100% sequence identity, such as about 81%; about 82%; about 83%; about84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%;about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%. Theproperty of substantial homology is closely related to specifichybridization. For example, a nucleic acid molecule is specificallyhybridizable when there is a sufficient degree of complementarity toavoid non-specific binding of the nucleic acid to non-target sequencesunder conditions where specific binding is desired, for example, understringent hybridization conditions.

In some instances “homologous” may be used to refer to the relationshipof a first gene to a second gene by descent from a common ancestral DNAsequence. In such instances, the term, homolog, indicates a relationshipbetween genes separated by the event of speciation (see ortholog) or tothe relationship between genes separated by the event of geneticduplication (see paralog). In other instances “homologous” may be usedto refer to the level of sequence identity between one or morepolynucleotide sequences, in such instances the one or morepolynucleotide sequences do not necessarily descend from a commonancestral DNA sequence. Those with skill in the art are aware of theinterchangeably of the term “homologous” and appreciate the properapplication of the term.

As used herein, the term “ortholog” (or “orthologous”) refers to a genein two or more species that has evolved from a common ancestralnucleotide sequence, and may retain the same function in the two or morespecies.

As used herein, the term “paralogous” refers to genes related byduplication within a genome. Orthologs retain the same function in thecourse of evolution, whereas paralogs evolve new functions, even ifthese new functions are unrelated to the original gene function.

As used herein, two nucleic acid sequence molecules are said to exhibit“complete complementarity” when every nucleotide of a sequence read inthe 5′ to 3′ direction is complementary to every nucleotide of the othersequence when read in the 3′ to 5′ direction. A nucleotide sequence thatis complementary to a reference nucleotide sequence will exhibit asequence identical to the reverse complement sequence of the referencenucleotide sequence. These terms and descriptions are well defined inthe art and are easily understood by those of ordinary skill in the art.

When determining the percentage of sequence identity between amino acidsequences, it is well-known by those of skill in the art that theidentity of the amino acid in a given position provided by an alignmentmay differ without affecting desired properties of the polypeptidescomprising the aligned sequences. In these instances, the percentsequence identity may be adjusted to account for similarity betweenconservatively substituted amino acids. These adjustments are well-knownand commonly used by those of skill in the art. See, e.g., Myers andMiller (1988) Computer Applications in Biosciences 4:11-7. Statisticalmethods are known in the art and can be used in analysis of theidentified 7,018 optimal genomic loci.

As an embodiment, the identified optimal genomic loci comprising 7,018individual optimal genomic loci sequences can be analyzed via anF-distribution test. In probability theory and statistics, theF-distribution is a continuous probability distribution. TheF-distribution test is a statistical significance test that has anF-distribution, and is used when comparing statistical models that havebeen fit to a data set, to identify the best-fitting model. AnF-distribution is a continuous probability distribution, and is alsoknown as Snedecor's F-distribution or the Fisher-Snedecor distribution.The F-distribution arises frequently as the null distribution of a teststatistic, most notably in the analysis of variance. The F-distributionis a right-skewed distribution. The F-distribution is an asymmetricdistribution that has a minimum value of 0, but no maximum value. Thecurve reaches a peak not far to the right of 0, and then graduallyapproaches the horizontal axis the larger the F value is. TheF-distribution approaches, but never quite touches the horizontal axis.It will be appreciated that in other embodiments, variations on thisequation, or indeed different equations, may be derived and used by theskilled person and are applicable to the analysis of 7,018 individualoptimal genomic loci sequences.

Operably linked: A first nucleotide sequence is “operably linked” with asecond nucleotide sequence when the first nucleotide sequence is in afunctional relationship with the second nucleotide sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.When recombinantly produced, operably linked nucleotide sequences aregenerally contiguous and, where necessary to join two protein-codingregions, in the same reading frame. However, nucleotide sequences neednot be contiguous to be operably linked.

The term, “operably linked,” when used in reference to a regulatorysequence and a coding sequence, means that the regulatory sequenceaffects the expression of the linked coding sequence. “Regulatorysequences,” “regulatory elements”, or “control elements,” refer tonucleotide sequences that influence the timing and level/amount oftranscription, RNA processing or stability, or translation of theassociated coding sequence. Regulatory sequences may include promoters;translation leader sequences; introns; enhancers; stem-loop structures;repressor binding sequences; termination sequences; polyadenylationrecognition sequences; etc. Particular regulatory sequences may belocated upstream and/or downstream of a coding sequence operably linkedthereto. Also, particular regulatory sequences operably linked to acoding sequence may be located on the associated complementary strand ofa double-stranded nucleic acid molecule.

When used in reference to two or more amino acid sequences, the term“operably linked” means that the first amino acid sequence is in afunctional relationship with at least one of the additional amino acidsequences.

The disclosed methods and compositions include fusion proteinscomprising a cleavage domain operably linked to a DNA-binding domain(e.g., a ZFP) in which the DNA-binding domain by binding to a sequencein the soybean optimal genomic locus directs the activity of thecleavage domain to the vicinity of the sequence and, hence, induces adouble stranded break in the optimal genomic locus. As set forthelsewhere in this disclosure, a zinc finger domain can be engineered tobind to virtually any desired sequence. Accordingly, one or moreDNA-binding domains can be engineered to bind to one or more sequencesin the optimal genomic locus. Expression of a fusion protein comprisinga DNA-binding domain and a cleavage domain in a cell, effects cleavageat or near the target site.

EMBODIMENTS

Targeting transgenes and transgene stacks to specific locations in thegenome of dicot plants, like a soybean plant, will improve the qualityof transgenic events, reduce costs associated with production oftransgenic events and provide new methods for making transgenic plantproducts such as sequential gene stacking. Overall, targeting trangenesto specific genomic sites is likely to be commercially beneficial.Significant advances have been made in the last few years towardsdevelopment of site-specific nucleases such as ZFNs, CRISPRs, and TALENsthat can facilitate addition of donor polynucleotides to pre-selectedsites in plant and other genomes. However, much less is known about theattributes of genomic sites that are suitable for targeting.Historically, non-essential genes and pathogen (viral) integration sitesin genomes have been used as loci for targeting. The number of suchsites in genomes is rather limiting and there is therefore a need foridentification and characterization of optimal genomic loci that can beused for targeting of donor polynucleotide sequences. In addition tobeing amenable to targeting, optimal genomic loci are expected to beneutral sites that can support transgene expression and breedingapplications.

Applicants have recognized that additional criteria are desirable forinsertion sites and have combined these criteria to identify and selectoptimal sites in the dicot genome, like the soybean genome, for theinsertion of exogenous sequences. For targeting purposes, the site ofselected insertion needs to be unique and in a non-repetitive region ofthe genome of a dicot plant, like a soybean plant. Likewise, the optimalgenomic site for insertion should possess minimal undesirable phenotypiceffects and be susceptible to recombination events to facilitateintrogression into agronomically elite lines using traditional breedingtechniques. In order to identify the genomic loci that meet the listedcriteria, the genome of a soybean plant was scanned using a customizedbioinformatics approach and genome scale datasets to identify novelgenomic loci possessing characteristics that are beneficial for theintegration of polynucleotide donor sequence and the subsequentexpression of an inserted coding sequence.

I. Identification of Nongenic Soybean Genomic Loci

In accordance with one embodiment a method is provided for identifyingoptimal nongenic soybean genomic sequence for insertion of exogenoussequences. The method comprises the steps of first identifying soybeangenomic sequences of at least 1 Kb in length that are hypomethylated. Inone embodiment the hypomethylated genomic sequence is 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11, 12, 13, 14, 15,16 or 17 Kb in length. In one embodiment the hypomethylated genomicsequence is about 1 to about 5.7 Kb in length and in a furtherembodiment is about 2 Kb in length. A sequence is consideredhypomethylated if it has less than 1% DNA methylation within thesequence. In one embodiment the methylation status is measured based onthe presence of 5-methylcytosine at one or more CpG dinucleotides, CHGor CHH trinucleotides within a selected soybean sequence, relative tothe amount of total cytosines found at corresponding CpG dinucleotides,CHG or CHH trinucleotides within a normal control DNA sample. CHHmethylation indicates a 5-methylcytosine followed by two nucleotidesthat many not be guanine and CHG methylation refers to a5-methylcytosine preceding an adenine, thymine or cytocine basedfollowed by guanine. More particularly, in one embodiment the selectedsoybean sequence has less than 1, 2 or 3 methylated nucleotides per 500nucleotides of the selected soybean sequence. In one embodiment theselected soybean sequence has less than one, two, or three5-methylcytosines at CpG dinucleotides per 500 nucleotides of theselected soybean sequence. In one embodiment the selected soybeansequence is 1 to 4 Kb in length and comprises a 1 Kb sequence devoid of5-methylcytosines. In one embodiment the selected soybean sequence is 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6, Kb in length and contains 1or 0 methylated nucleotides in its entire length. In one embodiment theselected soybean sequence is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or6, Kb in length and contains no 5-methylcytosines at CpG dinucleotideswithin in its entire length. In accordance with one embodiment themethylation of a selected soybean sequence may vary based on sourcetissue. In such embodiments the methylation levels used to determine ifa sequence is hypomethylated represents the average amount ofmethylation in the sequences isolated from two or more tissues (e.g.,from root and shoot).

In addition to the requirement that an optimal genomic site behypomethylated, the selected soybean sequence must also be nongenic.Accordingly, all hypomethylated genomic sequences are further screenedto eliminate hypomethylated sequences that contain a genic region. Thisincludes any open reading frames regardless of whether the transcriptencodes a protein. Hypomethylated genomic sequences that include genicregions, including any identifiable adjacent 5′ and 3′ non-codingnucleotide sequences involved in the regulation of expression of an openreading frame and any introns that may be present in the genic region,are excluded from the optimal nongenic soybean genomic locus of thepresent disclosure.

Optimal nongenic soybean genomic loci must also be sequences that havedemonstrated evidence of recombination. In one embodiment the selectedsoybean sequence must be one where at least one recombination event hasbeen detected between two markers flanking the selected soybean sequenceas detected using a high resolution marker dataset generated frommultiple mapping populations. In one embodiment the pair of markersflanking a 0.5, 1, 1.5 Mb dicot genomic sequence, such as a soybeangenomic sequence, comprising the selected soybean sequence are used tocalculate the recombinant frequency for the selected soybean sequence.Recombination frequencies between each pairs of markers (measured incentimorgan (cM)) to the genomic physical distance between the markers(in Mb)) must be greater than 0.0157 cM/Mb. In one embodiment therecombination frequency for a 1 Mb soybean genomic sequence comprisingthe selected soybean sequence ranges from about 0.01574 cM/Mb to about83.52 cM/Mb. In one embodiment an optimal genomic loci is one whererecombination events have been detected within the selected soybeansequence.

An optimal nongenic soybean genomic loci will also be a targetablesequence, i.e., a sequence that is relatively unique in the soybeangenome such that a gene targeted to the selected soybean sequence willonly insert in one location of the soybean genome. In one embodiment theentire length of the optimal genomic sequence shares less than 30%, 35%,or 40%, sequence identity with another sequence of similar lengthcontained in the soybean genome. Accordingly, in one embodiment theselected soybean sequence cannot comprise a 1 Kb sequence that sharemore than 25%, 30%, 35%, or 40% sequence identity with another 1 Kbsequence contained in the soybean genome. In a further embodiment theselected soybean sequence cannot comprise a 500 bp sequence that sharemore than 30%, 35%, or 40% sequence identity with another 500 bpsequence contained in the soybean genome. In one embodiment the selectedsoybean sequence cannot comprise a 1 Kb sequence that share more than40% sequence identity with another 1 Kb sequence contained in the genomeof a dicot plant, like a soybean plant.

An optimal nongenic soybean genomic loci will also be proximal to agenic region. More particularly, a selected soybean sequence must belocated in the vicinity of a genic region (e.g., a genic region must belocated within 40 Kb of genomic sequence flanking and contiguous witheither end of the selected soybean as found in the native genome). Inone embodiment a genic region is located within 10, 20, 30 or 40 Kb ofcontiguous genomic sequence located at either end of the selectedsoybean sequence as found in the native soybean genome. In oneembodiment two or more genic regions are located within 10, 20, 30 or 40Kb of contiguous genomic sequence flanking the two ends of the selectedsoybean sequence. In one embodiment 1-18 genic regions are locatedwithin 10, 20, 30 or 40 Kb of contiguous genomic sequence flanking thetwo ends of the selected soybean sequence. In one embodiment two or moregenic regions are located within a 20, 30 or 40 Kb genomic sequencecomprising the selected soybean sequence. In one embodiment 1-18 genicregions are located within a 40 Kb genomic sequence comprising theselected soybean sequence. In one embodiment the genic region locatedwithin a 10, 20, 30 or 40 Kb of contiguous genomic sequence flanking theselected soybean sequence comprises a known gene in the genome of adicot plant, such as a soybean plant.

In accordance with one embodiment a modified nongenic soybean genomicloci is provided wherein the loci is at least 1 Kb in length, isnongenic, comprises no methylated cytosine residues, has a recombinationfrequency of greater than 0.01574 cM/Mb over a 1 Mb genomic regionencompassing the soybean genomic loci and a 1 Kb sequence of the soybeangenomic loci shares less than 40% sequence identity with any other 1 Kbsequence contained in the dicot genome, wherein the nongenic soybeangenomic loci is modified by the insertion of a DNA of interest into thenongenic soybean genomic loci.

In accordance with one embodiment a method for identifying optimalnongenic dicot genomic loci, including for example, soybean genomic lociis provided. In some embodiments, the method first comprises screeningthe dicot genome to create a first pool of selected soybean sequencesthat have a minimal length of 1 Kb and are hypomethylated, optionallywherein the genomic sequence has less than 1% methylation, optionallywherein the genomic sequence is devoid of any methylated cytosineresidues. This first pool of selected soybean sequences can be furtherscreened to eliminate loci that do not meet the requirements for optimalnongenic soybean genomic loci. Dicot genomic sequences, such as thoseobtained from soybean, that encode dicot transcripts, share greater than40% or higher sequence identity with another sequence of similar length,do not exhibit evidence of recombination, and do not have a known openreading frame within 40 Kb of the selected soybean sequence, areeliminated from the first pool of sequences, leaving a second pool ofsequences that qualify as optimal nongenic soybean loci. In oneembodiment any selected soybean sequences that do not have a known dicotgene (i.e., a soybean gene), or a sequence comprising a 2 Kb upstreamand/or 1 Kb downstream region of a known dicot gene, within 40 Kb of oneend of said nongenic sequence are eliminated from the first pool ofsequences. In one embodiment any selected soybean sequences that do notcontain a known gene that expresses a protein within 40 Kb of theselected soybean sequence are eliminated. In one embodiment any selectedsoybean sequences that do not have a recombination frequency of greaterthan 0.01574 cM/Mb are eliminated.

Using these selection criteria applicants have identified select optimalgenomic loci of dicot, such as soybean, that serve as optimal nongenicsoybean genomic loci, the sequences of which are disclosed as SEQ ID NO:1-SEQ ID NO: 7,018. The present disclosure also encompasses naturalvariants or modified derivatives of the identified optimal nongenicsoybean genomic loci wherein the variant or derivative loci comprise asequence that differs from any sequence of SEQ ID NO: 1-SEQ ID NO: 7,018by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In one embodimentoptimal nongenic soybean genomic loci for use in accordance with thepresent disclosure comprise sequences selected from SEQ ID NO: 1-SEQ IDNO: 7,018 or sequences that share 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity with a sequence selected from SEQ IDNO: 1-SEQ ID NO: 7,018.

In another embodiment, dicot plants for use in accordance with thepresent disclosure comprise any plant selected from the group consistingof a soybean plant, a canola plant, a rape plant, a Brassica plant, acotton plant, and a sunflower plant. Examples of dicot plants that canbe used include, but are not limited to, canola, cotton, potato, quinoa,amaranth, buckwheat, safflower, soybean, sugarbeet, sunflower, canola,rape, tobacco, Arabidopsis, Brassica, and cotton.

In another embodiment, optimal nongenic soybean genomic loci for use inaccordance with the present disclosure comprise sequences selected fromsoybean plants. In a further embodiment, optimal nongenic soybeangenomic loci for use in accordance with the present disclosure comprisesequences selected from Glycine max inbreds. Accordingly, a Glycine maxinbred includes agronomically elite varieties thereof. In a subsequentembodiment, optimal nongenic soybean genomic loci for use in accordancewith the present disclosure comprise sequences selected fromtransformable soybean lines. In an embodiment, representativetransformable soybean lines include; Maverick, Williams82, MerrillJackPeking, Suzuyutaka, Fayette, Enrei, Mikawashima, WaseMidori, Jack,Leculus, Morocco, Serena, Maple prest, Thorne, Bert, Jungery, A3237,Williams, Williams79, AC Colibri, Hefeng 25, Dongnong 42, Hienong 37,Jilin 39, Jiyu 58, A3237, Kentucky Wonder, Minidoka, and derivativesthereof. One of skill in the art will appreciate that as a result ofphylogenetic divergence, various types of soybean lines do not containidentical genomic DNA sequences, and that polymorphisms or allelicvariation may be present within genomic sequences. In an embodiment, thepresent disclosure encompasses such polymorphism or allelic variationsof the identified optimal nongenic soybean genomic loci wherein thepolymorphisms or allelic variation comprise a sequence that differs fromany sequence with SEQ ID NO: 1-SEQ ID NO: 7,018 by 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 nucleotides. In a further embodiment, the present disclosureencompasses such polymorphisms or allelic variations of the identifiedoptimal nongenic soybean genomic loci wherein the sequences comprisingthe polymorphisms or allelic variation share 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity with any sequence of SEQ IDNO: 1-SEQ ID NO: 7,018.

The identified optimal genomic loci comprising 7,018 individualsequences can be categorized into various subgroupings by furtheranalysis using a multivariate analysis method. Application of anymultivariate analysis statistical programs is used to uncover the latentstructure (dimensions) of a set of variables. A number of differenttypes of multivariate algorithms can be used, for example the data setcan be analyzed using multiple regression analysis, logistic regressionanalysis, discriminate analysis, multivariate analysis of variance(MANOVA), factor analysis (including both common factor analysis, andprincipal component analysis), cluster analysis, multidimensionalscaling, correspondence analysis, conjoint analysis, canonical analysis,canonical correlation, and structural equation modeling.

In accordance with one embodiment the optimal nongenic soybean genomicloci are further analyzed using multivariate data analysis such asPrincipal Component Analysis (PCA). Only a brief description will begiven here, more information can be found in H. Martens, T. Naes,Multivariate Calibration, Wiley, N.Y., 1989. PCA evaluates theunderlying dimensionality (latent variables) of the data, and gives anoverview of the dominant patterns and major trends in the data. In oneembodiment, the optimal nongenic soybean genomic loci can be sorted intoclusters via a principal component analysis (PCA) statistical method.The PCA is a mathematical procedure that uses an orthogonaltransformation to convert a set of observations of possibly correlatedvariables into a set of values of linearly uncorrelated variables calledprincipal components. The number of principal components is less than orequal to the number of original variables. This transformation isdefined in such a way that the first principal component has the largestpossible variance (that is, accounts for as much of the variability inthe data as possible), and each succeeding component in turn has thehighest variance possible under the constraint that it be orthogonal to(i.e., uncorrelated with) the preceding components. Principal componentsare guaranteed to be independent if the data set is jointly normallydistributed. PCA is sensitive to the relative scaling of the originalvariables. Examples of the use of PCA to cluster a set of entities basedon features of the entities include; Ciampitti, I. et al., (2012) CropScience, 52(6); 2728-2742, Chemometrics: A Practical Guide, Kenneth R.Beebe, Randy J. Pell, and Mary Beth Seasholtz, Wiley-Interscience, 1edition, 1998, U.S. Pat. No. 8,385,662, and European Patent No.2,340,975.

In accordance with one embodiment a principal component analysis (PCA)was conducted on the 7,018 optimal soybean genomic loci using thefollowing 10 features for each identified optimal soybean genomic loci:

1. Length of the hypo-methylated region around the optimal soybeangenomic loci (OGL)

-   -   a. DNA methylation profiles of root and shoot tissues isolated        from a dicot plant, e.g., Glycine Max cultivar Williams82, were        constructed using a high throughput whole genome sequencing        approach. Extracted DNA was subjected to bisulphite treatment        that converts unmethylated cytosines to uracils, but does not        affect methylated cytosines, and then sequenced using Illumina        HiSeq technology (Krueger, F. et al. DNA methylome analysis        using short bisulfite sequencing data. Nature Methods 9, 145-151        (2012)). The raw sequencing reads were mapped to the dicot        reference sequence, e.g., Glycine max reference sequence, using        the Bismark™ mapping software (as described in Krueger F,        Andrews SR (2011) Bismark: a flexible aligner and methylation        caller for Bisulfite-Seq applications. (Bioinformatics 27:        1571-1572)). The length of the hypo-methylated region around        each of the OGLs was calculated using the described methylation        profiles.

2. Rate of Recombination in a 1 MB region around the OGL

-   -   a. For each OGL, a pair of markers on either side of the OGL,        within a 1 Mb window, was identified. Recombination frequencies        between each pairs of markers across the chromosome were        calculated based on the ratio of the genetic distance between        markers (in centimorgan (cM)) to the genomic physical distance        between the markers (in Mb).

3. Level of OGLsequence uniqueness

-   -   a. For each OGL, the nucleotide sequence of the OGL was scanned        against the genome of a dicot plant, e.g., soybean c.v.        Williams82 genome, using a BLAST based homology search. As these        OGL sequences are identified from the genome of a dicot plant,        e.g., soybean c.v. Williams82 genome, the first BLAST hit        identified through this search represents the OGL sequence        itself. The second BLAST hit for each OGL was identified and the        alignment coverage of the hit was used as a measure of        uniqueness of the OGL sequence within the dicot genome, e.g.,        soybean genome.

4. Distance from the OGL to the closest gene in its neighborhood

-   -   a. Gene annotation information and the location of known genes        in the dicot genome, e.g., soybean c.v. Williams82 genome, were        extracted from a known dicot genome database, e.g., Soybean        Genome Database (www.soybase.org). For each OGL, the closest        annotated gene in its upstream or downstream neighborhood was        identified and the distance between the OGL sequence and the        gene was measured (in bp).

5. GC % in the OGL neighborhood

-   -   a. For each OGL, the nucleotide sequence was analyzed to        estimate the number of Guanine and Cytosine bases present. This        count was represented as a percentage of the sequence length of        each OGL and provides a measure for GC %.

6. Number of genes in a 40 Kb neighborhood around the OGL

-   -   a. Gene annotation information and the location of known genes        in the dicot genome, e.g., soybean c.v. Williams82 genome, were        extracted from a known dicot genomic database, e.g., Soybean        Genome Database (www.soybase.org). For each OGL, a 40 Kb window        around the OGL was defined and the number of annotated genes        with locations overlapping this window was counted.

7. Average gene expression in a 40 Kb neighborhood around the OGL.

-   -   a. Transcript level expression of dicot genes, e.g., soybean        genes, was measured by analyzing transcriptome profiling data        generated from dicot plant tissues, e.g., soybean c.v.        Williams82 root and shoot tissues, using RNAseq technology. For        each OGL, annotated genes within the dicot genome, soybean c.v.        Williams82 genome, that were present in a 40 Kb neighborhood        around the OGL were identified. Expression levels for each of        the genes in the window were extracted from the transcriptome        profiles and an average gene expression level was calculated.

8. Level of Nucleosome occupancy around the OGL

-   -   a. Discerning the level of nucleosome occupancy for a particular        nucleotide sequence provides information about chromosomal        functions and the genomic context of the sequence. The NuPoP™        statistical package provides a user-friendly software tool for        predicting the nucleosome occupancy and the most probable        nucleosome positioning map for genomic sequences of any size        (Xi, L., Fondufe-Mittendor, Y., Xia, L., Flatow, J., Widom, J.        and Wang, J.-P., Predicting nucleosome positioning using a        duration Hidden Markov Model, BMC Bioinformatics, 2010,        doi:10.1186/1471-2105-11-346). For each OGL, the nucleotide        sequence was submitted to the NuPoP™ software and a nucleosome        occupancy score was calculated.

9. Relative location within the chromosome (proximity to centromere)

-   -   a. Information on position of the centromere in each of the        dicot chromosomes, e.g., soybean chromosomes, and the lengths of        the chromosome arms was extracted from a dicot genomic database,        e.g., Soybean Genome Databae (www.soybase.org). For each OGL,        the genomic distance from the OGL sequence to the centromere of        the chromosome that it is located on, is measured (in bp). The        relative location of a OGL within the chromosome is represented        as the ratio of its genomic distance to the centromere relative        to the length of the specific chromosomal arm that it lies on.

10. Number of OGLs in a 1 Mb region around the OGL

-   -   a. For each OGL, a 1 Mb genomic window around the OGL location        is defined and the number of OGLs, in the dicot 1 Kb OGL        dataset, whose genomic locations overlap with this window is        tallied.

The results or values for the score of the features and attributes ofeach optimal nongenic soybean genomic loci are further described inTable 3 of Example 2. The resulting dataset was used in the PCAstatistical method to cluster the 7,018 identified optimal nongenicsoybean genomic loci into clusters. During the clustering process, afterestimating the “p” principle components of the optimal genomic loci, theassignment of the optimal genomic loci to one of the 32 clustersproceeded in the “p” dimensional Euclidean space. Each of the “p” axeswas divided into “k” intervals. Optimal genomic loci assigned to thesame interval were grouped together to form clusters. Using thisanalysis, each PCA axis was divided into two intervals, which was chosenbased on a priori information regarding the number of clusters requiredfor experimental validation. All analysis and the visualization of theresulting clusters were carried out with the Molecular OperatingEnvironment™ (MOE) software from Chemical Computing Group Inc.(Montreal, Quebec, Canada). The PCA approach was used to cluster the setof 7,018 optimal soybean genomic loci into 32 distinct clusters based ontheir feature values, described above.

During the PCA process, five principal components (PC) were generated,with the top three PCs containing about 90% of the total variation inthe dataset (Table 4). These three PCs were used to graphicallyrepresent the 32 clusters in a three dimensional plot (see FIG. 1).After the clustering process, was completed, one representative optimalgenomic loci was chosen from each cluster. This was performed bychoosing a select optimal genomic locus, within each cluster, that wasclosest to the centroid of that cluster by computational methods (Table4). The chromosomal locations of the 32 representative optimal genomicloci are uniformly distributed among the soybean chromosomes as shown inFIG. 2.

In an embodiment an isolated or purified optimal nongenic soybeangenomic loci sequence is provided selected from any cluster described inTable 6 of Example 2. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 1. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 2. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 3. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 4. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 5. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 6. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 7. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 8. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 9. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 10. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 11. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 12. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 13. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 14. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 15. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 16. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 17. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 18. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 19. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 20. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 21. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 22. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 23. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 24. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 25. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 26. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 27. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 28. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 29. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 30. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 31. In one embodiment the isolated or purified optimalnongenic soybean genomic loci sequence is a genomic sequence selectedfrom cluster 32.

In accordance with one embodiment a modified optimal nongenic soybaengenomic loci is provided wherein the optimal nongenic soybean genomicloci has been modified and comprise one or more nucleotidesubstitutions, deletions or insertions. In one embodiment the optimalnongenic soybean genomic loci is modified by the insertion of a DNA ofinterest optionally accompanied with further nucleotide duplications,deletions or inversions of genomic loci sequence.

In an embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from any cluster described inTable 6 of Example 2. In one embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected fromcluster 1. In one embodiment the optimal nongenic soybean genomic locito be modified is a genomic sequence selected from cluster 2. In oneembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 3. In one embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 4. In one embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster5. In one embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 6. In oneembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 7. In one embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 8. In one embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster9. In one embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 10. In oneembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 11. In one embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 12. In one embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster13. In one embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 14. In oneembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 15. In one embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 16. In one embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster17. In one embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 18. In oneembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 19. In one embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 20. In one embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster21. In one embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 22. In oneembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 23. In one embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 24. In one embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster25. In one embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 26. In oneembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 27. In one embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 28. In one embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster29. In one embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 30. In oneembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 31. In one embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 32.

In a further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or 31. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,or 29. In a further embodiment the optimal nongenic soybean genomic locito be modified is a genomic sequence selected from cluster 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, or 27. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.In a further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, or 10. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, or 9. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, or 8. In a further embodimentthe optimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, or 7. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, or 6. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, or 5. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, or 4. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, or 3. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1 or 2.

In a further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodimentthe optimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31or 32. In a further embodiment the optimal nongenic soybean genomic locito be modified is a genomic sequence selected from cluster 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodimentthe optimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31or 32. In a further embodiment the optimal nongenic soybean genomic locito be modified is a genomic sequence selected from cluster 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24,25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26,27, 28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29,30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 29, 30, 31 or 32. In a further embodimentthe optimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31or 32. In a further embodiment the optimal nongenic soybean genomic locito be modified is a genomic sequence selected from cluster 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 32.

In a further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31 or 32. In a further embodiment the optimal nongenic soybean genomicloci to be modified is a genomic sequence selected from cluster 1, 2, 3,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31 or 32. In a further embodiment the optimal nongenic soybean genomicloci to be modified is a genomic sequence selected from cluster 1, 2, 3,4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31 or 32. In a further embodiment the optimal nongenic soybean genomicloci to be modified is a genomic sequence selected from cluster 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31 or 32. In a further embodiment the optimal nongenic soybean genomicloci to be modified is a genomic sequence selected from cluster 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27,28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30,31 or 32. In a further embodiment the optimal nongenic soybean genomicloci to be modified is a genomic sequence selected from cluster 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30,31 or 32. In a further embodiment the optimal nongenic soybean genomicloci to be modified is a genomic sequence selected from cluster 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 32.

In a further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31or 32. In a further embodiment the optimal nongenic soybean genomic locito be modified is a genomic sequence selected from cluster 1, 2, 3, 4,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 22, 23, 24, 25, 26, 27, 28,29, 30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 23,24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 26, 27, 28,29, 30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 32.

In a further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31 or 32. In a further embodiment the optimal nongenic soybeangenomic loci to be modified is a genomic sequence selected from cluster1, 2, 3, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment the optimalnongenic soybean genomic loci to be modified is a genomic sequenceselected from cluster 1, 2, 3, 4, 5, 6, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a furtherembodiment the optimal nongenic soybean genomic loci to be modified is agenomic sequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Ina further embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31 or 32. In a further embodiment the optimal nongenic soybean genomicloci to be modified is a genomic sequence selected from cluster 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 27, 28, 29, 30, 31 or 32. In afurther embodiment the optimal nongenic soybean genomic loci to bemodified is a genomic sequence selected from cluster 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 27, 28, 29, 30,31 or 32. In a further embodiment the optimal nongenic soybean genomicloci to be modified is a genomic sequence selected from cluster 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 30, 31 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, or 27. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 9, 10, 11, 12, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodimentthe optimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 15, 16, 17,18, 25, 26, 27, 28, 29, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 21, 22, 23, 24, 30, 31 or 32. In a further embodiment theoptimal nongenic soybean genomic loci to be modified is a genomicsequence selected from cluster 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30 or 32. In a further embodiment the optimal nongenicsoybean genomic loci to be modified is a genomic sequence selected fromcluster 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31.

In one embodiment the optimal nongenic soybean genomic loci is selectedfrom the genomic sequences of soy_ogl_2474 (SEQ ID NO: 1), soy_ogl_768(SEQ ID NO: 506), soy_ogl_2063 (SEQ ID NO: 2063), soy_ogl_1906 (SEQ IDNO: 1029), soy_ogl_1112 (SEQ ID NO: 1112), soy_ogl_3574 (SEQ ID NO:1452), soy_ogl_2581 (SEQ ID NO: 1662), soy_ogl_3481 (SEQ ID NO: 1869),soy_ogl_1016 (SEQ ID NO: 2071), soy_ogl_937 (SEQ ID NO: 2481),soy_ogl_6684 (SEQ ID NO: 2614), soy_ogl_6801 (SEQ ID NO: 2874),soy_ogl_6636 (SEQ ID NO: 2970), soy_ogl_4665 (SEQ ID NO: 3508),soy_ogl_3399 (SEQ ID NO: 3676), soy_ogl_4222 (SEQ ID NO: 3993),soy_ogl_2543 (SEQ ID NO: 4050), soy_ogl_275 (SEQ ID NO: 4106),soy_ogl_598 (SEQ ID NO: 4496), soy_ogl_1894 (SEQ ID NO: 4622),soy_ogl_5454 (SEQ ID NO: 4875), soy_ogl_6838 (SEQ ID NO: 4888),soy_ogl_4779 (SEQ ID NO: 5063), soy_ogl_3333 (SEQ ID NO: 5122),soy_ogl_2546 (SEQ ID NO: 5520), soy_ogl_796 (SEQ ID NO: 5687),soy_ogl_873 (SEQ ID NO: 6087), soy_ogl_5475 (SEQ ID NO: 6321),soy_ogl_2115 (SEQ ID NO: 6520), soy_ogl_2518 (SEQ ID NO: 6574),soy_ogl_5551 (SEQ ID NO: 6775), and soy_ogl_4563 (SEQ ID NO: 6859).

In one embodiment the optimal nongenic soybean genomic loci is selectedfrom the genomic sequences of soy_ogl_308 (SEQ ID NO: 43), soy_ogl_307(SEQ ID NO: 566), soy_ogl_2063 (SEQ ID NO: 748), soy_ogl_1906 (SEQ IDNO: 1029), soy_ogl_262 (SEQ ID NO: 1376), soy_ogl_5227 (SEQ ID NO:1461), soy_ogl_4074 (SEQ ID NO: 1867), soy_ogl_3481 (SEQ ID NO: 1869),soy_ogl_1016 (SEQ ID NO: 2071), soy_ogl_937 (SEQ ID NO: 2481),soy_ogl_5109 (SEQ ID NO: 2639), soy_ogl_6801 (SEQ ID NO: 2874),soy_ogl_6636 (SEQ ID NO: 2970), soy_ogl_4665 (SEQ ID NO: 3508),soy_ogl_6189 (SEQ ID NO: 3682), soy_ogl_4222 (SEQ ID NO: 3993),soy_ogl_2543 (SEQ ID NO: 4050), soy_ogl_310 (SEQ ID NO: 4326),soy_ogl_2353 (SEQ ID NO: 4593), soy_ogl_1894 (SEQ ID NO: 4622),soy_ogl_3669 (SEQ ID NO: 4879), soy_ogl_3218 (SEQ ID NO: 4932),soy_ogl_5689 (SEQ ID NO: 5102), soy_ogl_3333 (SEQ ID NO: 5122),soy_ogl_2546 (SEQ ID NO: 5520), soy_ogl_1208 (SEQ ID NO: 5698),soy_ogl_873 (SEQ ID NO: 6087), soy_ogl_5957 (SEQ ID NO: 6515),soy_ogl_4846 (SEQ ID NO: 6571), soy_ogl_3818 (SEQ ID NO: 6586),soy_ogl_5551 (SEQ ID NO: 6775), soy_ogl_7 (SEQ ID NO: 6935), soy_OGL_684(SEQ ID NO: 47), soy_OGL_682 (SEQ ID NO: 2101), soy_OGL_685 (SEQ ID NO:48), soy_OGL_1423 (SEQ ID NO: 639), soy_OGL_1434 (SEQ ID NO: 137),soy_OGL_4625 (SEQ ID NO: 76), and soy_OGL_6362 (SEQ ID NO: 440).

In one embodiment the optimal nongenic soybean genomic loci is targetedwith a DNA of interest, wherein the DNA of interest integrates within orproximal to the zinc finger nuclease target sites. In accordance with anembodiment, exemplary zinc finger target sites of optimal maize selectgenomic loci are provided in Table 8. In accordance with an embodiment,integration of a DNA of interest occurs within or proximal to theexemplary target sites of: SEQ ID NO: 7363 and SEQ ID NO: 7364, SEQ IDNO: 7365 and SEQ ID NO: 7366, SEQ ID NO: 7367 and SEQ ID NO: 7368, SEQID NO: 7369 and SEQ ID NO: 7370, SEQ ID NO: 7371 and SEQ ID NO: 7372,SEQ ID NO: 7373 and SEQ ID NO: 7374, SEQ ID NO: 7375 and SEQ ID NO:7376, SEQ ID NO: 7377 and SEQ ID NO: 7378, SEQ ID NO: 7379 and SEQ IDNO: 7380, SEQ ID NO: 7381 and SEQ ID NO: 7382, SEQ ID NO: 7383 and SEQID NO: 7384, SEQ ID NO: 7385 and SEQ ID NO: 7386, SEQ ID NO: 7387 andSEQ ID NO: 7388, SEQ ID NO: 7389 and SEQ ID NO: 7390, SEQ ID NO: 7391and SEQ ID NO: 7392, SEQ ID NO: 7393 and SEQ ID NO: 7394, SEQ ID NO:7395 and SEQ ID NO: 7396, SEQ ID NO: 7397 and SEQ ID NO: 7398, SEQ IDNO: 7399 and SEQ ID NO: 7400, SEQ ID NO: 7401 and SEQ ID NO: 7402, SEQID NO: 7403 and SEQ ID NO: 7404, SEQ ID NO: 7405 and SEQ ID NO: 7406,SEQ ID NO: 7407 and SEQ ID NO: 7408, SEQ ID NO: 7409 and SEQ ID NO:7410, SEQ ID NO: 7411 and SEQ ID NO: 7412, SEQ ID NO: 7413 and SEQ IDNO: 7414, SEQ ID NO: 7415 and SEQ ID NO: 7416, SEQ ID NO: 7417 and SEQID NO: 7418, SEQ ID NO: 7419 and SEQ ID NO: 7420, SEQ ID NO: 7421 andSEQ ID NO: 7422, SEQ ID NO: 7423 and SEQ ID NO: 7424, SEQ ID NO: 7425and SEQ ID NO: 7426.

In accordance with an embodiment, the zinc finger nuclease binds to thezinc finger target site and cleaves the unique soybean genomicpolynucleotide target sites, whereupon the DNA of interest integrateswithin or proximal to the soybean genomic polynucleotide target sites.In an embodiment, integration of the DNA of interest within the zincfinger target site may result with rearrangements. In accordance withone embodiment, the rearrangements may comprise deletions, insertions,inversions, and repeats. In an embodiment, integration of the DNA ofinterest occurs proximal to the zinc finger target site. According to anaspect of the embodiment, the integration of the DNA is proximal to thezinc finger target site and may integrate within 2 Kb, 1.75 Kb, 1.5 Kb,1.25 Kb, 1.0 Kb, 0.75 Kb, 0.5 Kb, or 0.25 Kb to the zinc finger targetsite. Insertion within a genomic region proximal to the zinc fingertarget site is known in the art, see US Patent Pub No. 2010/0257638 A1(herein incorporated by reference in its entirety).

In accordance with one embodiment the selected nongenic sequencecomprises the following characteristics:

a) the nongenic sequence does not contain greater than 1% DNAmethylation within the sequence;

b) the nongenic sequence has a relative location value from 0.211 to0.976 ratio of genomic distance from a soybean chromosomal centromere;

c) the nongenic sequence has a guanine/cytosine percent content range of25.62 to 43.76%; and,

d) the nongenic sequence is from about 1 Kb to about 4.4 Kb in length.

II. Recombinant Derivatives of Identified Optimal Nongenic SoybeanGenomic Loci

In accordance with one embodiment, after having identified a genomicloci of a dicot plant, such as a soybean plant, as a highly desirablelocation for inserting polynucleotide donor sequences, one or morenucleic acids of interest can be inserted into the identified genomiclocus. In one embodiment the nucleic acid of interest comprisesexogenous gene sequences or other desirable polynucleotide donorsequences. In another embodiment, after having identified a genomic lociof a dicot plant, such as a soybean plant, as a highly desirablelocation for inserting polynucleotide donor sequences, one or morenucleic acids of interest of the optimal nongenic soybean genomic locican optionally be deleted, excised or removed with the subsequentintegration of the DNA of interest into the identified genomic locus. Inone embodiment the insertion of a nucleic acid of interest into theoptimal nongenic soybean genomic loci comprises removal, deletion, orexcision of the exogenous gene sequences or other desirablepolynucleotide donor sequences.

The present disclosure further relates to methods and compositions fortargeted integration into the select soybean genomic locus using ZFNsand a polynucleotide donor construct. The methods for inserting anucleic acid sequence of interest into the optimal nongenic soybeangenomic loci, unless otherwise indicated, use conventional techniques inmolecular biology, biochemistry, chromatin structure and analysis, cellculture, recombinant DNA and related fields as are within the skill ofthe art. These techniques are fully explained in the literature. See,for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL,Second edition, Cold Spring Harbor Laboratory Press, 1989 and Thirdedition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, New York, 1987 and periodic updates; the seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998;METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Was sarman and A. P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Methods for Nucleic Acid Insertion into the Soybean Genome Any of thewell known procedures for introducing polynucleotide donor sequences andnuclease sequences as a DNA construct into host cells may be used inaccordance with the present disclosure. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, PEG,electroporation, ultrasonic methods (e.g., sonoporation), liposomes,microinjection, naked DNA, plasmid vectors, viral vectors, both episomaland integrative, and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). It isonly necessary that the particular nucleic acid insertion procedure usedbe capable, of successfully introducing at least one gene into the hostcell capable of expressing the protein of choice.

As noted above, DNA constructs may be introduced into the genome of adesired plant species by a variety of conventional techniques. Forreviews of such techniques see, for example, Weissbach & WeissbachMethods for Plant Molecular Biology (1988, Academic Press, N.Y.) SectionVIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988,2d Ed.), Blackie, London, Ch. 7-9. A DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation and microinjection of plant cell protoplasts, byagitation with silicon carbide fibers (See, e.g., U.S. Pat. Nos.5,302,523 and 5,464,765), or the DNA constructs can be introduceddirectly to plant tissue using biolistic methods, such as DNA particlebombardment (see, e.g., Klein et al. (1987) Nature 327:70-73).Alternatively, the DNA construct can be introduced into the plant cellvia nanoparticle transformation (see, e.g., US Patent Publication No.20090104700, which is incorporated herein by reference in its entirety).Alternatively, the DNA constructs may be combined with suitable T-DNAborder/flanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. Agrobacterium tumefaciens-mediatedtransformation techniques, including disarming and use of binaryvectors, are well described in the scientific literature. See, forexample Horsch et al. (1984) Science 233:496-498, and Fraley et al.(1983) Proc. Nat'l. Acad. Sci. USA 80:4803.

In addition, gene transfer may be achieved using non-Agrobacteriumbacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virusand cassava vein mosaic virus and/or tobacco mosaic virus, See, e.g.,Chung et al. (2006) Trends Plant Sci. 11(1):1-4. The virulence functionsof the Agrobacterium tumefaciens host will direct the insertion of aT-strand containing the construct and adjacent marker into the plantcell DNA when the cell is infected by the bacteria using binary T DNAvector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivationprocedure (Horsch et al. (1985) Science 227:1229-1231). Generally, theAgrobacterium transformation system is used to engineer dicotyledonousplants (Bevan et al. (1982) Ann. Rev. Genet. 16:357-384; Rogers et al.(1986) Methods Enzymol. 118:627-641). The Agrobacterium transformationsystem may also be used to transform, as well as transfer, DNA tomonocotyledonous plants and plant cells. See U.S. Pat. No. 5,591,616;Hernalsteen et al. (1984) EMBO J. 3:3039-3041; Hooykass-Van Slogteren etal. (1984) Nature 311:763-764; Grimsley et al. (1987) Nature325:1677-179; Boulton et al. (1989) Plant Mol. Biol. 12:31-40; and Gouldet al. (1991) Plant Physiol. 95:426-434.

Alternative gene transfer and transformation methods include, but arenot limited to, protoplast transformation through calcium-, polyethyleneglycol (PEG)- or electroporation-mediated uptake of naked DNA (seePaszkowski et al. (1984) EMBO J. 3:2717-2722, Potrykus et al. (1985)Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad.Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) andelectroporation of plant tissues (D'Halluin et al. (1992) Plant Cell4:1495-1505). Additional methods for plant cell transformation includemicroinjection, silicon carbide mediated DNA uptake (Kaeppler et al.(1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment(see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; andGordon-Kamm et al. (1990) Plant Cell 2:603-618).

In one embodiment a nucleic acid of interest introduced into a host cellfor targeted insertion into the genome comprises homologous flankingsequences on one or both ends of the targeted nucleic acid of interest.In such an embodiment, the homologous flanking sequences containsufficient levels of sequence identity to a dicot genomic sequence, suchas a genomic sequence from soybean, to support homologous recombinationbetween it and the genomic sequence to which it bears homology.Approximately 25, 50, 100, 200, 500, 750, 1000, 1500, or 2000nucleotides, or more of sequence identity, ranging from 70% to 100%,between a donor and a genomic sequence (or any integral value between 10and 200 nucleotides, or more) will support homologous recombinationtherebetween.

In another embodiment the targeted nucleic acid of interest lackshomologous flanking sequences, and the targeted nucleic acid of interestshares low to very low levels of sequence identity with a genomicsequence.

In other embodiments of targeted recombination and/or replacement and/oralteration of a sequence in a region of interest in cellular chromatin,a chromosomal sequence is altered by homologous recombination with anexogenous “donor” nucleotide sequence. Such homologous recombination isstimulated by the presence of a double-stranded break in cellularchromatin, if sequences homologous to the region of the break arepresent. Double-strand breaks in cellular chromatin can also stimulatecellular mechanisms of non-homologous end joining. In any of the methodsdescribed herein, the first nucleotide sequence (the “donor sequence”)can contain sequences that are homologous, but not identical, to genomicsequences in the region of interest, thereby stimulating homologousrecombination to insert a non-identical sequence in the region ofinterest. Thus, in certain embodiments, portions of the donor sequencethat are homologous to sequences in the region of interest exhibitbetween about 80, 85, 90, 95, 97.5, to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs.

In certain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50 to 2,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 2,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the region of interest, and isinserted into the genome by non-homologous recombination mechanisms.

In accordance with one embodiment a zinc finger nuclease (ZFN) is usedto introduce a double strand break in a targeted genomic locus tofacilitate the insertion of a nucleic acid of interest. Selection of atarget site within the selected genomic locus for binding by a zincfinger domain can be accomplished, for example, according to the methodsdisclosed in U.S. Pat. No. 6,453,242, the disclosure of which isincorporated herein, that also discloses methods for designing zincfinger proteins (ZFPs) to bind to a selected sequence. It will be clearto those skilled in the art that simple visual inspection of anucleotide sequence can also be used for selection of a target site.Accordingly, any means for target site selection can be used in themethods described herein.

For ZFP DNA-binding domains, target sites are generally composed of aplurality of adjacent target subsites. A target subsite refers to thesequence, usually either a nucleotide triplet or a nucleotide quadrupletwhich may overlap by one nucleotide with an adjacent quadruplet that isbound by an individual zinc finger. See, for example, WO 02/077227, thedisclosure of which is incorporated herein. A target site generally hasa length of at least 9 nucleotides and, accordingly, is bound by a zincfinger binding domain comprising at least three zinc fingers. Howeverbinding of, for example, a 4-finger binding domain to a 12-nucleotidetarget site, a 5-finger binding domain to a 15-nucleotide target site ora 6-finger binding domain to an 18-nucleotide target site, is alsopossible. As will be apparent, binding of larger binding domains (e.g.,7-, 8-, 9-finger and more) to longer target sites is also consistentwith the subject disclosure.

In accordance with one embodiment, it is not necessary for a target siteto be a multiple of three nucleotides. In cases in which cross-strandinteractions occur (see, e.g., U.S. Pat. No. 6,453,242 and WO02/077227), one or more of the individual zinc fingers of a multi-fingerbinding domain can bind to overlapping quadruplet subsites. As a result,a three-finger protein can bind a 10-nucleotide sequence, wherein thetenth nucleotide is part of a quadruplet bound by a terminal finger, afour-finger protein can bind a 13-nucleotide sequence, wherein thethirteenth nucleotide is part of a quadruplet bound by a terminalfinger, etc.

The length and nature of amino acid linker sequences between individualzinc fingers in a multi-finger binding domain also affects binding to atarget sequence. For example, the presence of a so-called “non-canonicallinker,” “long linker” or “structured linker” between adjacent zincfingers in a multi-finger binding domain can allow those fingers to bindsubsites which are not immediately adjacent. Non-limiting examples ofsuch linkers are described, for example, in U.S. Pat. No. 6,479,626 andWO 01/53480. Accordingly, one or more subsites, in a target site for azinc finger binding domain, can be separated from each other by 1, 2, 3,4, 5 or more nucleotides. One nonlimiting example would be a four-fingerbinding domain that binds to a 13-nucleotide target site comprising, insequence, two contiguous 3-nucleotide subsites, an interveningnucleotide, and two contiguous triplet subsites.

While DNA-binding polypeptides identified from proteins that exist innature typically bind to a discrete nucleotide sequence or motif (e.g.,a consensus recognition sequence), methods exist and are known in theart for modifying many such DNA-binding polypeptides to recognize adifferent nucleotide sequence or motif. DNA-binding polypeptidesinclude, for example and without limitation: zinc finger DNA-bindingdomains; leucine zippers; UPA DNA-binding domains; GAL4; TAL; LexA; aTet repressor; LacR; and a steroid hormone receptor.

In some examples, a DNA-binding polypeptide is a zinc finger. Individualzinc finger motifs can be designed to target and bind specifically toany of a large range of DNA sites. Canonical Cys₂H is₂ (as well asnon-canonical Cys₃His) zinc finger polypeptides bind DNA by inserting anα-helix into the major groove of the target DNA double helix.Recognition of DNA by a zinc finger is modular; each finger contactsprimarily three consecutive base pairs in the target, and a few keyresidues in the polypeptide mediate recognition. By including multiplezinc finger DNA-binding domains in a targeting endonuclease, theDNA-binding specificity of the targeting endonuclease may be furtherincreased (and hence the specificity of any gene regulatory effectsconferred thereby may also be increased). See, e.g., Urnov et al. (2005)Nature 435:646-51. Thus, one or more zinc finger DNA-bindingpolypeptides may be engineered and utilized such that a targetingendonuclease introduced into a host cell interacts with a DNA sequencethat is unique within the genome of the host cell. Preferably, the zincfinger protein is non-naturally occurring in that it is engineered tobind to a target site of choice. See, for example, Beerli et al. (2002)Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal etal. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr.Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261;6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317;7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent PublicationNos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated hereinby reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. In addition, theDNA-binding specificity of homing endonucleases and meganucleases can beengineered to bind non-natural target sites. See, for example, Chevalieret al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic AcidsRes. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques etal. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128.

As another alternative, the DNA-binding domain may be derived from aleucine zipper protein. Leucine zippers are a class of proteins that areinvolved in protein-protein interactions in many eukaryotic regulatoryproteins that are important transcription factors associated with geneexpression. The leucine zipper refers to a common structural motifshared in these transcriptional factors across several kingdomsincluding animals, plants, yeasts, etc. The leucine zipper is formed bytwo polypeptides (homodimer or heterodimer) that bind to specific DNAsequences in a manner where the leucine residues are evenly spacedthrough an α-helix, such that the leucine residues of the twopolypeptides end up on the same face of the helix. The DNA bindingspecificity of leucine zippers can be utilized in the DNA-bindingdomains disclosed herein.

In some embodiments, the DNA-binding domain is an engineered domain froma TAL effector derived from the plant pathogen Xanthomonas (see, Milleret al. (2011) Nature Biotechnology 29(2):143-8; Boch et al, (2009)Science 29 Oct. 2009 (10.1126/science.117881) and Moscou and Bogdanove,(2009) Science 29 Oct. 2009 (10.1126/science.1178817; and U.S. PatentPublication Nos. 20110239315, 20110145940 and 20110301073).

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system is a recentlyengineered nuclease system based on a bacterial system that can be usedfor genome engineering. It is based on part of the adaptive immuneresponse of many bacteria and Archea. When a virus or plasmid invades abacterium, segments of the invader's DNA are converted into CRISPR RNAs(crRNA) by the ‘immune’ response. This crRNA then associates, through aregion of partial complementarity, with another type of RNA calledtracrRNA to guide the Cas9 nuclease to a region homologous to the crRNAin the target DNA called a “protospacer”. Cas9 cleaves the DNA togenerate blunt ends at the DSB at sites specified by a 20-nucleotideguide sequence contained within the crRNA transcript. Cas9 requires boththe crRNA and the tracrRNA for site specific DNA recognition andcleavage. This system has now been engineered such that the crRNA andtracrRNA can be combined into one molecule (the “single guide RNA”), andthe crRNA equivalent portion of the single guide RNA can be engineeredto guide the Cas9 nuclease to target any desired sequence (see Jinek etal (2012) Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471,and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cas system canbe engineered to create a double-stranded break (DSB) at a desiredtarget in a genome, and repair of the DSB can be influenced by the useof repair inhibitors to cause an increase in error prone repair.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. Suitable derivatives of a Caspolypeptide or a fragment thereof include but are not limited tomutants, fusions, covalent modifications of Cas protein or a fragmentthereof. Cas protein, which includes Cas protein or a fragment thereof,as well as derivatives of Cas protein or a fragment thereof, may beobtainable from a cell or synthesized chemically or by a combination ofthese two procedures. The cell may be a cell that naturally produces Casprotein, or a cell that naturally produces Cas protein and isgenetically engineered to produce the endogenous Cas protein at a higherexpression level or to produce a Cas protein from an exogenouslyintroduced nucleic acid, which nucleic acid encodes a Cas that is sameor different from the endogenous Cas. In some case, the cell does notnaturally produce Cas protein and is genetically engineered to produce aCas protein. The Cas protein is deployed in mammalian cells (andputatively within plant cells) by co-expressing the Cas nuclease withguide RNA. Two forms of guide RNAs can be used to facilitateCas-mediated genome cleavage as disclosed in Le Cong, F., et al., (2013)Science 339(6121):819-823.

In other embodiments, the DNA-binding domain may be associated with acleavage (nuclease) domain. For example, homing endonucleases may bemodified in their DNA-binding specificity while retaining nucleasefunction. In addition, zinc finger proteins may also be fused to acleavage domain to form a zinc finger nuclease (ZFN). The cleavagedomain portion of the fusion proteins disclosed herein can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). Nonlimiting examples of homing endonucleases and meganucleases includeI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. Seealso U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al.(1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin(1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. One or more of these enzymes (or functionalfragments thereof) can be used as a source of cleavage domains andcleavage half-domains.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type 11S) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-FokI fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-FokI fusionsare provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 2007/014275, incorporated by reference herein in itsentirety.

To enhance cleavage specificity, cleavage domains may also be modified.In certain embodiments, variants of the cleavage half-domain areemployed these variants minimize or prevent homodimerization of thecleavage half-domains. Non-limiting examples of such modified cleavagehalf-domains are described in detail in WO 2007/014275, incorporated byreference in its entirety herein. In certain embodiments, the cleavagedomain comprises an engineered cleavage half-domain (also referred to asdimerization domain mutants) that minimize or prevent homodimerization.Such embodiments are known to those of skill the art and described forexample in U.S. Patent Publication Nos. 20050064474; 20060188987;20070305346 and 20080131962, the disclosures of all of which areincorporated by reference in their entireties herein. Amino acidresidues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496,498, 499, 500, 531, 534, 537, and 538 of FokI are all targets forinfluencing dimerization of the FokI cleavage half-domains.

Additional engineered cleavage half-domains of FokI that form obligateheterodimers can also be used in the ZFNs described herein. Exemplaryengineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499. In one embodiment, a mutation at 490 replaces Glu(E) with Lys (K); the mutation at 538 replaces Iso (I) with Lys (K); themutation at 486 replaced Gln (O) with Glu (E); and the mutation atposition 499 replaces Iso (I) with Lys (K). Specifically, the engineeredcleavage half-domains described herein were prepared by mutatingpositions 490 (E→K) and 538 (I→K) in one cleavage half-domain to producean engineered cleavage half-domain designated “E490K:I538K” and bymutating positions 486 (Q→E) and 499 (I→L) in another cleavagehalf-domain to produce an engineered cleavage half-domain designated“Q486E:I499L”. The engineered cleavage half-domains described herein areobligate heterodimer mutants in which aberrant cleavage is minimized orabolished. See, e.g., U.S. Patent Publication No. 2008/0131962, thedisclosure of which is incorporated by reference in its entirety for allpurposes. In certain embodiments, the engineered cleavage half-domaincomprises mutations at positions 486, 499 and 496 (numbered relative towild-type FokI), for instance mutations that replace the wild type Gln(O) residue at position 486 with a Glu (E) residue, the wild type Iso(I) residue at position 499 with a Leu (L) residue and the wild-type Asn(N) residue at position 496 with an Asp (D) or Glu (E) residue (alsoreferred to as a “ELD” and “ELE” domains, respectively). In otherembodiments, the engineered cleavage half-domain comprises mutations atpositions 490, 538 and 537 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Glu (E) residue atposition 490 with a Lys (K) residue, the wild type Iso (I) residue atposition 538 with a Lys (K) residue, and the wild-type H is (H) residueat position 537 with a Lys (K) residue or a Arg (R) residue (alsoreferred to as “KKK” and “KKR” domains, respectively). In otherembodiments, the engineered cleavage half-domain comprises mutations atpositions 490 and 537 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Glu (E) residue atposition 490 with a Lys (K) residue and the wild-type H is (H) residueat position 537 with a Lys (K) residue or a Arg (R) residue (alsoreferred to as “KIK” and “KIR” domains, respectively). (See US PatentPublication No. 20110201055). In other embodiments, the engineeredcleavage half domain comprises the “Sharkey” and/or “Sharkey” mutations(see Guo et al, (2010) J. Mol. Biol. 400(1):96-107).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474; 20080131962; and 20110201055.Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and20090068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014,275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter, for example the galactokinasepromoter which is activated (de-repressed) in the presence of raffinoseand/or galactose and repressed in presence of glucose.

Distance between target sites refers to the number of nucleotides ornucleotide pairs intervening between two target sites as measured fromthe edges of the sequences nearest each other. In certain embodiments inwhich cleavage depends on the binding of two zinc finger domain/cleavagehalf-domain fusion molecules to separate target sites, the two targetsites can be on opposite DNA strands. In other embodiments, both targetsites are on the same DNA strand. For targeted integration into theoptimal genomic locus, one or more ZFPs are engineered to bind a targetsite at or near the predetermined cleavage site, and a fusion proteincomprising the engineered DNA-binding domain and a cleavage domain isexpressed in the cell. Upon binding of the zinc finger portion of thefusion protein to the target site, the DNA is cleaved, preferably via adouble-stranded break, near the target site by the cleavage domain.

The presence of a double-stranded break in the optimal genomic locusfacilitates integration of exogenous sequences via homologousrecombination. Thus, in one embodiment the polynucleotide comprising thenucleic acid sequence of interest to be inserted into the targetedgenomic locus will include one or more regions of homology with thetargeted genomic locus to facilitate homologous recombination.

In addition to the fusion molecules described herein, targetedreplacement of a selected genomic sequence also involves theintroduction of a donor sequence. The polynucleotide donor sequence canbe introduced into the cell prior to, concurrently with, or subsequentto, expression of the fusion protein(s). In one embodiment the donorpolynucleotide contains sufficient homology to the optimal genomic locusto support homologous recombination between it and the optimal genomiclocus genomic sequence to which it bears homology. Approximately 25, 50,100, 200, 500, 750, 1,000, 1,500, 2,000 nucleotides or more of sequencehomology between a donor and a genomic sequence, or any integral valuebetween 10 and 2,000 nucleotides or more, will support homologousrecombination. In certain embodiments, the homology arms are less than1,000 basepairs in length. In other embodiments, the homology arms areless than 750 base pairs in length. In one embodiment the donorpolynucleotide sequences can comprise a vector molecule containingsequences that are not homologous to the region of interest in cellularchromatin. A donor polynucleotide molecule can contain several,discontinuous regions of homology to cellular chromatin. For example,for targeted insertion of sequences not normally present in a region ofinterest, said sequences can be present in a donor nucleic acid moleculeand flanked by regions of homology to sequence in the region ofinterest. The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. See, e.g., U.S. Patent Publication Nos. 20100047805, 20110281361,20110207221 and U.S. application Ser. No. 13/889,162. If introduced inlinear form, the ends of the donor sequence can be protected (e.g., fromexonucleolytic degradation) by methods known to those of skill in theart. For example, one or more dideoxynucleotide residues are added tothe 3′ terminus of a linear molecule and/or self-complementaryoligonucleotides are ligated to one or both ends. See, for example,Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls etal. (1996) Science 272:886-889. Additional methods for protectingexogenous polynucleotides from degradation include, but are not limitedto, addition of terminal amino group(s) and the use of modifiedinternucleotide linkages such as, for example, phosphorothioates,phosphoramidates, and O-methyl ribose or deoxyribose residues.

In accordance with one embodiment a method of preparing a transgenicdicot plant, such as a soybean plant, is provided wherein a DNA ofinterest has been inserted into an optimal nongenic soybean genomiclocus. The method comprises the steps of:

a. selecting an optimal nongenic soybean locus as a target for insertionof the nucleic acid of interest;

b. introducing a site specific nuclease into a dicot plant cell, such asa soybean plant cell, wherein the site specific nuclease cleaves thenongenic sequence;

c. introducing the DNA of interest into the plant cell; and

d. selecting transgenic plant cells comprising the DNA of interesttargeted to said nongenic sequence.

In accordance with one embodiment a method of preparing a transgenicdicot protoplast cell, like a soybean protoplast cell, is providedwherein a DNA of interest has been inserted into an optimal nongenicsoybean genomic locus. The method comprises the steps of:

a. selecting an optimal nongenic soybean locus as a target for insertionof the nucleic acid of interest;

b. introducing a site specific nuclease into a dicot protoplast cell,like a soybean protoplast cell, wherein the site specific nucleasecleaves the nongenic sequence;

c. introducing the DNA of interest into the dicot protoplast cell, likea soybean protoplast cell; and

d. selecting the transgenic dicot protoplast cell, like a soybeanprotoplast cell, comprising the DNA of interest targeted to saidnongenic sequence.

In one embodiment the site specific nuclease is selected from the groupconsisting of a Zinc Finger nuclease, a CRISPR nuclease, a TALENnuclease, or a meganuclease, and more particularly in one embodiment thesite specific nuclease is a Zinc Finger nuclease. In accordance with oneembodiment the DNA of interest is integrated within said nongenicsequence via a homology directed repair integration method.Alternatively, in some embodiments the DNA of interest is integratedwithin said nongenic sequence via a non-homologous end joiningintegration method. In additional embodiments, the DNA of interest isintegrated within said nongenic sequence via a previously undescribedintegration method. In one embodiment the method comprises selecting aoptimal nongenic soybean genomic locus for targeted insertion of a DNAof interest that has 2, 3, 4, 5, 6, 7, or 8 of the followingcharacteristics:

a. the nongenic sequence is at least 1 Kb in length and does not containgreater than 1% DNA methylation within the sequence

b. the nongenic sequence exhibits a 0.01574 to 83.52 cM/Mb rate ofrecombination within the dicot genome, like a soybean genome;

c. the nongenic sequence exhibits a 0 to 0.494 level of nucleosomeoccupancy of the dicot genome, like a soybean genome;

d. the nongenic sequence shares less than 40% sequence identity with anyother sequence contained in the dicot genome, like a soybean genome;

e. the nongenic sequence has a relative location value from 0 to 0.99682ratio of genomic distance from a dicot chromosomal centromere, likesoybean;

f. the nongenic sequence has a guanine/cytosine percent content range of14.4 to 45.9%;

g. the nongenic sequence is located proximally to a genic sequence; and,

h. a 1 Mb region of dicot genomic sequence, like a soybean genomicsequence, comprising said nongenic sequence comprises one or moreadditional nongenic sequences. In one embodiment the optimal nongenicsoybean locus is selected from a loci of cluster 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 2, 3, 4, 5, 6, 7, 8, 9, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31 or 32.

Delivery

The donor molecules disclosed herein are integrated into a genome of acell via targeted, homology-independent and/or homology-dependentmethods. For such targeted integration, the genome is cleaved at adesired location (or locations) using a nuclease, for example, a fusionbetween a DNA-binding domain (e.g., zinc finger binding domain, CRISPRor TAL effector domain is engineered to bind a target site at or nearthe predetermined cleavage site) and nuclease domain (e.g., cleavagedomain or cleavage half-domain). In certain embodiments, two fusionproteins, each comprising a DNA-binding domain and a cleavagehalf-domain, are expressed in a cell, and bind to target sites which arejuxtaposed in such a way that a functional cleavage domain isreconstituted and DNA is cleaved in the vicinity of the target sites. Inone embodiment, cleavage occurs between the target sites of the twoDNA-binding domains. One or both of the DNA-binding domains can beengineered. See, also, U.S. Pat. No. 7,888,121; U.S. Patent Publication20050064474 and International Patent Publications WO05/084190,WO05/014791 and WO 03/080809.

The nucleases as described herein can be introduced as polypeptidesand/or polynucleotides. For example, two polynucleotides, eachcomprising sequences encoding one of the aforementioned polypeptides,can be introduced into a cell, and when the polypeptides are expressedand each binds to its target sequence, cleavage occurs at or near thetarget sequence. Alternatively, a single polynucleotide comprisingsequences encoding both fusion polypeptides is introduced into a cell.Polynucleotides can be DNA, RNA or any modified forms or analogues orDNA and/or RNA.

Following the introduction of a double-stranded break in the region ofinterest, the transgene is integrated into the region of interest in atargeted manner via non-homology dependent methods (e.g., non-homologousend joining (NHEJ)) following linearization of a double-stranded donormolecule as described herein. The double-stranded donor is preferablylinearized in vivo with a nuclease, for example one or more of the sameor different nucleases that are used to introduce the double-strandedbreak in the genome. Synchronized cleavage of the chromosome and thedonor in the cell may limit donor DNA degradation (as compared tolinearization of the donor molecule prior to introduction into thecell). The nuclease target sites used for linearization of the donorpreferably do not disrupt the transgene(s) sequence(s).

The transgene may be integrated into the genome in the directionexpected by simple ligation of the nuclease overhangs (designated“forward” or “AB” orientation) or in the alternate direction (designated“reverse” or “BA” orientation). In certain embodiments, the transgene isintegrated following accurate ligation of the donor and chromosomeoverhangs. In other embodiments, integration of the transgene in eitherthe BA or AB orientation results in deletion of several nucleotides.

Through the application of techniques such as these, the cells ofvirtually any species may be stably transformed. In some embodiments,transforming DNA is integrated into the genome of the host cell. In thecase of multicellular species, transgenic cells may be regenerated intoa transgenic organism. Any of these techniques may be used to produce atransgenic plant, for example, comprising one or more donorpolynucleotide acid sequences in the genome of the transgenic plant.

The delivery of nucleic acids may be introduced into a plant cell inembodiments of the invention by any method known to those of skill inthe art, including, for example and without limitation: bytransformation of protoplasts (See, e.g., U.S. Pat. No. 5,508,184); bydesiccation/inhibition-mediated DNA uptake (See, e.g., Potrykus et al.(1985) Mol. Gen. Genet. 199:183-8); by electroporation (See, e.g., U.S.Pat. No. 5,384,253); by agitation with silicon carbide fibers (See,e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765); by Agrobacterium-mediatedtransformation (See, e.g., U.S. Pat. Nos. 5,563,055, 5,591,616,5,693,512, 5,824,877, 5,981,840, and 6,384,301); by acceleration ofDNA-coated particles (See, e.g., U.S. Pat. Nos. 5,015,580, 5,550,318,5,538,880, 6,160,208, 6,399,861, and 6,403,865) and by Nanoparticles,nanocarriers and cell penetrating peptides (WO201126644A2;WO2009046384A1; WO2008148223A1) in the methods to deliver DNA, RNA,Peptides and/or proteins or combinations of nucleic acids and peptidesinto plant cells.

The most widely-utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria that genetically transform plant cells. The T_(i) andR_(i) plasmids of A. tumefaciens and A. rhizogenes, respectively, carrygenes responsible for genetic transformation of the plant. The T_(i)(tumor-inducing)-plasmids contain a large segment, known as T-DNA, whichis transferred to transformed plants. Another segment of the T_(i)plasmid, the vir region, is responsible for T-DNA transfer. The T-DNAregion is bordered by left-hand and right-hand borders that are eachcomposed of terminal repeated nucleotide sequences. In some modifiedbinary vectors, the tumor-inducing genes have been deleted, and thefunctions of the vir region are utilized to transfer foreign DNAbordered by the T-DNA border sequences. The T-region may also contain,for example, a selectable marker for efficient recovery of transgenicplants and cells, and a multiple cloning site for inserting sequencesfor transfer such as a nucleic acid encoding a fusion protein of theinvention.

Thus, in some embodiments, a plant transformation vector is derived froma T_(i) plasmid of A. tumefaciens (See, e.g., U.S. Pat. Nos. 4,536,475,4,693,977, 4,886,937, and 5,501,967; and European Patent EP 0 122 791)or a R_(i) plasmid of A. rhizogenes. Additional plant transformationvectors include, for example and without limitation, those described byHerrera-Estrella et al. (1983) Nature 303:209-13; Bevan et al. (1983),supra; Klee et al. (1985) Bio/Technol. 3:637-42; and in European PatentEP 0 120 516, and those derived from any of the foregoing. Otherbacteria, such as Sinorhizobium, Rhizobium, and Mesorhizobium, thatnaturally interact with plants can be modified to mediate gene transferto a number of diverse plants. These plant-associated symbiotic bacteriacan be made competent for gene transfer by acquisition of both adisarmed T_(i) plasmid and a suitable binary vector.

The Nucleic Acid of Interest

The polynucleotide donor sequences for targeted insertion into a genomiclocus of a dicot plant, like a soybean plant, typically range in lengthfrom about 10 to about 5,000 nucleotides. However, nucleotidessubstantially longer, up to 20,000 nucleotides can be used, includingsequences of about 5, 6, 7, 8, 9, 10, 11 and 12 Kb in length.Additionally, donor sequences can comprise a vector molecule containingsequences that are not homologous to the replaced region. In oneembodiment the nucleic acid of interest will include one or more regionsthat share homology with the targeted genomic loci. Generally, thehomologous region(s) of the nucleic acid sequence of interest will haveat least 50% sequence identity to a genomic sequence with whichrecombination is desired. In certain embodiments, the homologousregion(s) of the nucleic acid of interest shares 60%, 70%, 80%, 90%,95%, 98%, 99%, or 99.9% sequence identity with sequences located in thetargeted genomic locus. However, any value between 1% and 100% sequenceidentity can be present, depending upon the length of the nucleic acidof interest.

A nucleic acid of interest can contain several, discontinuous regions ofsequence sharing relatively high sequence identity to cellularchromatin. For example, for targeted insertion of sequences not normallypresent in a targeted genomic locus, the unique sequences can be presentin a donor nucleic acid molecule and flanked by regions of sequencesthat share a relatively high sequence identity to a sequence present inthe targeted genomic locus.

A nucleic acid of interest can also be inserted into a targeted genomiclocus to serve as a reservoir for later use. For example, a firstnucleic acid sequence comprising sequences homologous to a nongenicregion of the genome of a dicot plant, like a soybean plant, butcontaining a nucleic acid of interest (optionally encoding a ZFN underthe control of an inducible promoter), may be inserted in a targetedgenomic locus. Next, a second nucleic acid sequence is introduced intothe cell to induce the insertion of a DNA of interest into an optimalnongenic genomic locus of a dicot plant, like a soybean plant. Eitherthe first nucleic acid sequence comprises a ZFN specific to the optimalnongenic soybean genomic locus and the second nucleic acid sequencecomprises the DNA sequence of interest, or vice versa. In one embodimentthe ZFN will cleave both the optimal nongenic soybean genomic locus andthe nucleic acid of interest. The resulting double stranded break in thegenome can then become the integration site for the nucleic acid ofinterest released from the optimal genomic locus. Alternatively,expression of a ZFN already located in the genome can be induced afterintroduction of the DNA of interest to induce a double stranded break inthe genome that can then become the integration site for the introducednucleic acid of interest. In this way, the efficiency of targetedintegration of a DNA of interest at any region of interest may beimproved since the method does not rely on simultaneous uptake of boththe nucleic acids encoding the ZFNs and the DNA of interest.

A nucleic acid of interest can also be inserted into an optimal nongenicsoybean genomic locus to serve as a target site for subsequentinsertions. For example, a nucleic acid of interest comprised of DNAsequences that contain recognition sites for additional ZFN designs maybe inserted into the locus. Subsequently, additional ZFN designs may begenerated and expressed in cells such that the original nucleic acid ofinterest is cleaved and modified by repair or homologous recombination.In this way, reiterative integrations of nucleic acid of interests mayoccur at the optimal nongenic genomic locus of a dicot plant, like asoybean plant.

Exemplary exogenous sequences that can be inserted into an optimalnongenic soybean genomic locus include, but are not limited to, anypolypeptide coding sequence (e.g., cDNAs), promoter, enhancer and otherregulatory sequences (e.g., interfering RNA sequences, shRNA expressioncassettes, epitope tags, marker genes, cleavage enzyme recognition sitesand various types of expression constructs. Such sequences can bereadily obtained using standard molecular biological techniques(cloning, synthesis, etc.) and/or are commercially available.

To express ZFNs, sequences encoding the fusion proteins are typicallysubcloned into an expression vector that contains a promoter to directtranscription. Suitable prokaryotic and eukaryotic promoters are wellknown in the art and described, e.g., in Sambrook et al., MolecularCloning, A Laboratory Manual (2nd ed. 1989; 3.sup.rd ed., 2001);Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); andCurrent Protocols in Molecular Biology (Ausubel et al., supra. Bacterialexpression systems for expressing the ZFNs are available in, e.g., E.coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235(1983)). Kits for such expression systems are commercially available.Eukaryotic expression systems for mammalian cells, yeast, and insectcells are well known by those of skill in the art and are alsocommercially available.

The particular expression vector used to transport the genetic materialinto the cell is selected with regard to the intended use of the fusionproteins, e.g., expression in plants, animals, bacteria, fungus,protozoa, etc. (see expression vectors described below). Standardbacterial and animal expression vectors are known in the art and aredescribed in detail, for example, U.S. Patent Publication 20050064474A1and International Patent Publications WO05/084190, WO05/014791 andWO03/080809.

Standard transfection methods can be used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofprotein, which can then be purified using standard techniques (see,e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide toProtein Purification, in Methods in Enzymology, vol. 182 (Deutscher,ed., 1990)). Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds., 1983).

The disclosed methods and compositions can be used to insertpolynucleotide donor sequences into a predetermined location such as oneof the optimal nongenic soybean genomic loci. This is useful inasmuch asexpression of an introduced transgene into the soybean genome dependscritically on its integration site. Accordingly, genes encodingherbicide tolerance, insect resistance, nutrients, antibiotics ortherapeutic molecules can be inserted, by targeted recombination.

In one embodiment the nucleic acid of interest is combined or “stacked”with gene encoding sequences that provide additional resistance ortolerance to glyphosate or another herbicide, and/or provides resistanceto select insects or diseases and/or nutritional enhancements, and/orimproved agronomic characteristics, and/or proteins or other productsuseful in feed, food, industrial, pharmaceutical or other uses. The“stacking” of two or more nucleic acid sequences of interest within aplant genome can be accomplished, for example, via conventional plantbreeding using two or more events, transformation of a plant with aconstruct which contains the sequences of interest, re-transformation ofa transgenic plant, or addition of new traits through targetedintegration via homologous recombination.

Such polynucleotide donor nucleotide sequences of interest include, butare not limited to, those examples provided below:

1. Genes or Coding Sequence (e.g. iRNA) That Confer Resistance to Pestsor Disease

(A) Plant Disease Resistance Genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. Examples of such genes include, the tomato Cf-9 genefor resistance to Cladosporium fulvum (Jones et al., 1994 Science266:789), tomato Pto gene, which encodes a protein kinase, forresistance to Pseudomonas syringae pv. tomato (Martin et al., 1993Science 262:1432), and Arabidopsis RSSP2 gene for resistance toPseudomonas syringae (Mindrinos et al., 1994 Cell 78:1089).

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon, such as, a nucleotide sequence ofa Bt δ-endotoxin gene (Geiser et al., 1986 Gene 48:109), and avegetative insecticidal (VIP) gene (see, e.g., Estruch et al. (1996)Proc. Natl. Acad. Sci. 93:5389-94). Moreover, DNA molecules encodingδ-endotoxin genes can be purchased from American Type Culture Collection(Rockville, Md.), under ATCC accession numbers 40098, 67136, 31995 and31998.

(C) A lectin, such as, nucleotide sequences of several Clivia miniatamannose-binding lectin genes (Van Damme et al., 1994 Plant Molec. Biol.24:825).

(D) A vitamin binding protein, such as avidin and avidin homologs whichare useful as larvicides against insect pests. See U.S. Pat. No.5,659,026.

(E) An enzyme inhibitor, e.g., a protease inhibitor or an amylaseinhibitor. Examples of such genes include a rice cysteine proteinaseinhibitor (Abe et al., 1987 J. Biol. Chem. 262:16793), a tobaccoproteinase inhibitor I (Huub et al., 1993 Plant Molec. Biol. 21:985),and an α-amylase inhibitor (Sumitani et al., 1993 Biosci. Biotech.Biochem. 57:1243).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof, such as baculovirus expression of clonedjuvenile hormone esterase, an inactivator of juvenile hormone (Hammocket al., 1990 Nature 344:458).

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest (J. Biol. Chem. 269:9).Examples of such genes include an insect diuretic hormone receptor(Regan, 1994), an allostatin identified in Diploptera punctata (Pratt,1989), and insect-specific, paralytic neurotoxins (U.S. Pat. No.5,266,361).

(H) An insect-specific venom produced in nature by a snake, a wasp,etc., such as a scorpion insectotoxic peptide (Pang, 1992 Gene 116:165).

(I) An enzyme responsible for a hyperaccumulation of monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, anuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. Examples ofsuch genes include, a callas gene (PCT published applicationWO93/02197), chitinase-encoding sequences (which can be obtained, forexample, from the ATCC under accession numbers 3999637 and 67152),tobacco hookworm chitinase (Kramer et al., 1993 Insect Molec. Biol.23:691), and parsley ubi-4-2 polyubiquitin gene (Kawalleck et al., 1993Plant Molec. Biol. 21:673).

(K) A molecule that stimulates signal transduction. Examples of suchmolecules include nucleotide sequences for mung bean calmodulin cDNAclones (Botella et al., 1994 Plant Molec. Biol. 24:757) and a nucleotidesequence of a soybean calmodulin cDNA clone (Griess et al., 1994 PlantPhysiol. 104:1467).

(L) A hydrophobic moment peptide. See U.S. Pat. Nos. 5,659,026 and5,607,914; the latter teaches synthetic antimicrobial peptides thatconfer disease resistance.

(M) A membrane permease, a channel former or a channel blocker, such asa cecropin-β lytic peptide analog (Jaynes et al., 1993 Plant Sci. 89:43)which renders transgenic tobacco plants resistant to Pseudomonassolanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. See,for example, Beachy et al. (1990) Ann. Rev. Phytopathol. 28:451.

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Forexample, Taylor et al. (1994) Abstract #497, Seventh Int'l. Symposium onMolecular Plant-Microbe Interactions shows enzymatic inactivation intransgenic tobacco via production of single-chain antibody fragments.

(P) A virus-specific antibody. See, for example, Tavladoraki et al.(1993) Nature 266:469, which shows that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(O) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase (Lamb et al., 1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.(1992 Plant J. 2:367).

(R) A developmental-arrestive protein produced in nature by a plant,such as the barley ribosome-inactivating gene that provides an increasedresistance to fungal disease (Longemann et al., 1992). Bio/Technology10:3305.

(S) RNA interference, in which an RNA molecule is used to inhibitexpression of a target gene. An RNA molecule in one example is partiallyor fully double stranded, which triggers a silencing response, resultingin cleavage of dsRNA into small interfering RNAs, which are thenincorporated into a targeting complex that destroys homologous mRNAs.See, e.g., Fire et al., U.S. Pat. No. 6,506,559; Graham et al. U.S. Pat.No. 6,573,099.

2. Genes That Confer Resistance to a Herbicide

(A) Genes encoding resistance or tolerance to a herbicide that inhibitsthe growing point or meristem, such as an imidazalinone, sulfonanilideor sulfonylurea herbicide. Exemplary genes in this category code formutant acetolactate synthase (ALS) (Lee et al., 1988 EMBO J. 7:1241)also known as acetohydroxyacid synthase (AHAS) enzyme (Miki et al., 1990Theor. Appl. Genet. 80:449).

(B) One or more additional genes encoding resistance or tolerance toglyphosate imparted by mutant EPSP synthase and aroA genes, or throughmetabolic inactivation by genes such as DGT-28, 2mEPSPS, GAT (glyphosateacetyltransferase) or GOX (glyphosate oxidase) and other phosphonocompounds such as glufosinate (pat,bar, and dsm-2 genes), andaryloxyphenoxypropionic acids and cyclohexanediones (ACCase inhibitorencoding genes). See, for example, U.S. Pat. No. 4,940,835, whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC Accession Number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061. Europeanpatent application No. 0 333 033 and U.S. Pat. No. 4,975,374 disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricinacetyl-transferase gene is provided inEuropean application No. 0 242 246. De Greef et al. (1989)Bio/Technology 7:61 describes the production of transgenic plants thatexpress chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance toaryloxyphenoxypropionic acids and cyclohexanediones, such as sethoxydimand haloxyfop, are the Accl-S1, Accl-S2 and Accl-S3 genes described byMarshall et al. (1992) Theor. Appl. Genet. 83:435.

(C) Genes encoding resistance or tolerance to a herbicide that inhibitsphotosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla et al. (1991) Plant Cell 3:169describe the use of plasmids encoding mutant psbA genes to transformChlamydomonas. Nucleotide sequences for nitrilase genes are disclosed inU.S. Pat. No. 4,810,648, and DNA molecules containing these genes areavailable under ATCC accession numbers 53435, 67441 and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al. (1992) Biochem. J. 285:173.

(D) Genes encoding resistance or tolerance to a herbicide that bind tohydroxyphenylpyruvate dioxygenases (HPPD), enzymes which catalyze thereaction in which para-hydroxyphenylpyruvate (HPP) is transformed intohomogentisate. This includes herbicides such as isoxazoles (EP418175,EP470856, EP487352, EP527036, EP560482, EP682659, U.S. Pat. No.5,424,276), in particular isoxaflutole, which is a selective herbicidefor soybean, diketonitriles (EP496630, EP496631), in particular2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3 phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3Cl2-phenyl)propane-1,3-dione,triketones (EP625505, EP625508, U.S. Pat. No. 5,506,195), in particularsulcotrione, and pyrazolinates. A gene that produces an overabundance ofHPPD in plants can provide tolerance or resistance to such herbicides,including, for example, genes described in U.S. Pat. Nos. 6,268,549 and6,245,968 and U.S. Patent Application Publication No. 20030066102.

(E) Genes encoding resistance or tolerance to phenoxy auxin herbicides,such as 2,4-dichlorophenoxyacetic acid (2,4-D) and which may also conferresistance or tolerance to aryloxyphenoxypropionate (AOPP) herbicides.Examples of such genes include the α-ketoglutarate-dependent dioxygenaseenzyme (aad-1) gene, described in U.S. Pat. No. 7,838,733.

(F) Genes encoding resistance or tolerance to phenoxy auxin herbicides,such as 2,4-dichlorophenoxyacetic acid (2,4-D) and which may also conferresistance or tolerance to pyridyloxy auxin herbicides, such asfluoroxypyr or triclopyr. Examples of such genes include theα-ketoglutarate-dependent dioxygenase enzyme gene (aad-12), described inWO 2007/053482 A2.

(G) Genes encoding resistance or tolerance to dicamba (see, e.g., U.S.Patent Publication No. 20030135879).

(H) Genes providing resistance or tolerance to herbicides that inhibitprotoporphyrinogen oxidase (PPO) (see U.S. Pat. No. 5,767,373).

(I) Genes providing resistance or tolerance to triazine herbicides (suchas atrazine) and urea derivatives (such as diuron) herbicides which bindto core proteins of photosystem II reaction centers (PS II) (SeeBrussian et al., (1989) EMBO J. 1989, 8(4): 1237-1245.

3. Genes That Confer or Contribute to a Value-Added Trait

(A) Modified fatty acid metabolism, for example, by transforming soybeanor Brassica with an antisense gene or stearoyl-ACP desaturase toincrease stearic acid content of the plant (Knultzon et al., 1992) Proc.Nat. Acad. Sci. USA 89:2624.

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene, such as the Aspergillusniger phytase gene (Van Hartingsveldt et al., 1993 Gene 127:87),enhances breakdown of phytate, adding more free phosphate to thetransformed plant.

(2) A gene could be introduced that reduces phytate content. In dicots,this, for example, could be accomplished by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor soybean mutants characterized by low levels of phytic acid (Raboy etal., 1990 Maydica 35:383).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. Examples of such enzymes include,Streptococcus mucus fructosyltransferase gene (Shiroza et al., 1988) J.Bacteriol. 170:810, Bacillus subtilis levansucrase gene (Steinmetz etal., 1985 Mol. Gen. Genel. 200:220), Bacillus lichenifonnis α-amylase(Pen et al., 1992 Bio/Technology 10:292), tomato invertase genes (Elliotet al., 1993), barley amylase gene (Sogaard et al., 1993 J. Biol. Chem.268:22480), and soybean endosperm starch branching enzyme II (Fisher etal., 1993 Plant Physiol. 102:10450).

III. Recombinant Constructs

As disclosed herein the present disclosure provides recombinant genomicsequences comprising an optimal nongenic soybean genomic sequence of atleast 1 Kb and a DNA of interest, wherein the inserted DNA of interestis inserted into said nongenic sequence. In one embodiment the DNA ofinterest is an analytical domain, a gene or coding sequence (e.g. iRNA)that confers resistance to pests or disease, genes that conferresistance to a herbicide or genes that confer or contribute to avalue-added trait, and the optimal nongenic soybean genomic sequencecomprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following characteristics:

a. the nongenic sequence is about 1 Kb to about 5.7 Kb in length anddoes not contain a methylated polynucleotide;

b. the nongenic sequence exhibits a 0.01574 to 83.52 cM/Mb rate ofrecombination within the genome of a dicot plant, like a soybean plant;

c. the nongenic sequence exhibits a 0 to 0.494 level of nucleosomeoccupancy of the dicot genome, like a soybean genome;

d. the nongenic sequence shares less than 40% sequence identity with anyother sequence contained in the dicot genome, like a soybean genome;

e. the nongenic sequence has a relative location value from 0 to 0.99682ratio of genomic distance from a dicot chromosomal centromere, like asoybean chromosomal center;

f. the nongenic sequence has a guanine/cytosine percent content range of14.4 to 45.9%;

g. the nongenic sequence is located proximally to an genic sequence,comprising a known or predicted dicot coding sequence, such as a soybeancoding sequence, within 40 Kb of contiguous genomic DNA comprising thenative nongenic sequence; and,

h. the nongenic sequence is located in a 1 Mb region of dicot genomicsequence, such as a genomic sequence, that comprises at least a secondnongenic sequence.

In one embodiment the optimal nongenic soybean genomic sequence isfurther characterized as having a genic region comprisings 1 to 18 knownor predicted soybean coding sequence within 40 Kb of contiguous genomicDNA comprising the native nongenic sequence.

In one embodiment the optimal nongenic soybean locus is selected from aloci of cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 3, 4, 5, 6, 7, 8,9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.

IV. Transgenic Plants

Transgenic plants comprising the recombinant optimal nongenic soybeanloci are also provided in accordance with one embodiment of the presentdisclosure. Such transgenic plants can be prepared using techniquesknown to those skilled in the art.

A transformed dicot cell, callus, tissue or plant (i.e., a soybean cell,callus, tissue or plant) may be identified and isolated by selecting orscreening the engineered plant material for traits encoded by the markergenes present on the transforming DNA. For instance, selection can beperformed by growing the engineered plant material on media containingan inhibitory amount of the antibiotic or herbicide to which thetransforming gene construct confers resistance. Further, transformedcells can also be identified by screening for the activities of anyvisible marker genes (e.g., the yellow fluorescence protein, greenfluorescence protein, red fluorescence protein, beta-glucuronidase,luciferase, B or C1 genes) that may be present on the recombinantnucleic acid constructs. Such selection and screening methodologies arewell known to those skilled in the art.

Physical and biochemical methods also may be used to identify plant orplant cell transformants containing inserted gene constructs. Thesemethods include but are not limited to: 1) Southern analysis or PCRamplification for detecting and determining the structure of therecombinant DNA insert; 2) Northern blot, S1 RNase protection,primer-extension or reverse transcriptase-PCR amplification fordetecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, Western blot techniques, immunoprecipitation, orenzyme-linked immunoassays (ELISA), where the gene construct productsare proteins. Additional techniques, such as in situ hybridization,enzyme staining, and immunostaining, also may be used to detect thepresence or expression of the recombinant construct in specific plantorgans and tissues. The methods for doing all these assays are wellknown to those skilled in the art.

Effects of gene manipulation using the methods disclosed herein can beobserved by, for example, Northern blots of the RNA (e.g., mRNA)isolated from the tissues of interest. Typically, if the mRNA is presentor the amount of mRNA has increased, it can be assumed that thecorresponding transgene is being expressed. Other methods of measuringgene and/or encoded polypeptide activity can be used. Different types ofenzymatic assays can be used, depending on the substrate used and themethod of detecting the increase or decrease of a reaction product orby-product. In addition, the levels of polypeptide expressed can bemeasured immunochemically, i.e., ELISA, RIA, EIA and other antibodybased assays well known to those of skill in the art, such as byelectrophoretic detection assays (either with staining or westernblotting). As one non-limiting example, the detection of the AAD-12(aryloxyalkanoate dioxygenase; see WO 2011/066360) and PAT(phosphinothricin-N-acetyl-transferase (PAT)) proteins using an ELISAassay is described in U.S. Patent Publication No. 20090093366 which isherein incorporated by reference in its entirety. The transgene may beselectively expressed in some tissues of the plant or at somedevelopmental stages, or the transgene may be expressed in substantiallyall plant tissues, substantially along its entire life cycle. However,any combinatorial expression mode is also applicable.

One of skill in the art will recognize that after the exogenouspolynucleotide donor sequence is stably incorporated in transgenicplants and confirmed to be operable, it can be introduced into otherplants by sexual crossing. Any of a number of standard breedingtechniques can be used, depending upon the species to be crossed.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above wherein the seed has the transgene or gene construct.The present disclosure further encompasses the progeny, clones, celllines or cells of the transgenic plants described above wherein theprogeny, clone, cell line or cell has the transgene or gene constructinserted into an optimal genomic loci.

Transformed plant cells which are produced by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans, et al., “Protoplasts Isolation andCulture” in Handbook of Plant Cell Culture, pp. 124-176, MacmillianPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, pollens,embryos or parts thereof. Such regeneration techniques are describedgenerally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486.

A transgenic plant or plant material comprising a nucleotide sequenceencoding a polypeptide may in some embodiments exhibit one or more ofthe following characteristics: expression of the polypeptide in a cellof the plant; expression of a portion of the polypeptide in a plastid ofa cell of the plant; import of the polypeptide from the cytosol of acell of the plant into a plastid of the cell; plastid-specificexpression of the polypeptide in a cell of the plant; and/orlocalization of the polypeptide in a cell of the plant. Such a plant mayadditionally have one or more desirable traits other than expression ofthe encoded polypeptide. Such traits may include, for example:resistance to insects, other pests, and disease-causing agents;tolerances to herbicides; enhanced stability, yield, or shelf-life;environmental tolerances; pharmaceutical production; industrial productproduction; and nutritional enhancements.

In accordance with one embodiment a transgenic dicot protoplast (i.e., asoybean protoplast) is provided comprising a recombinant optimalnongenic soybean locus. More particularly, a dicot protoplast, such as asoybean protoplast, is provided comprising a DNA of interest insertedinto an optimal nongenic soybean genomic loci of the dicot protoplast(i.e, a soybean protoplast), wherein said nongenic soybean genomic lociis about 1 Kb to about 5.7 Kb in length and lacks any methylatednucleotides. In one embodiment the transgenic dicot protoplast (i.e., atransgenic soybean protoplat), comprises a DNA of interest inserted intothe optimal nongenic soybean genomic locus wherein the DNA of interestcomprises an analytical domain, and/or an open reading frame. In oneembodiment the inserted DNA of interest encodes a peptide and in afurther embodiment the DNA of interest comprises at least one geneexpression cassette comprising a transgene.

In accordance with one embodiment a transgenic dicot plant, dicot plantpart, or dicot plant cell (i.e., a transgenic soybean plant, soybeanplant part, or soybean plant cell) is provided comprising a recombinantoptimal nongenic soybean locus. More particularly, a dicot plant, dicotplant part, or dicot plant cell (i.e., a soybean plant, soybean plantpart, or soybean plant cell) is provided comprising a DNA of interestinserted into an optimal nongenic soybean genomic loci of the dicotplant, dicot plant part, or dicot plant cell (i.e., a soybean plant,soybean plant part, or soybean plant cell), wherein said nongenicsoybean genomic loci is about 1 Kb to about 5.7 Kb in length and lacksany methylated nucleotides. In one embodiment the transgenic dicotplant, dicot plant part, or dicot plant cell (i.e., a transgenic soybeanplant, soybean plant part, or soybean plant cell) comprises a DNA ofinterest inserted into the optimal nongenic soybean genomic locuswherein the DNA of interest comprises an analytical domain, and/or anopen reading frame. In one embodiment the inserted DNA of interestencodes a peptide and in a further embodiment the DNA of interestcomprises at least one gene expression cassette comprising a transgene.

In accordance with embodiment 1a recombinant sequence is provided,wherein a nongenic soybean genomic sequence of at least 1 Kb, saidnongenic sequence being hypomethylated, targetable, located proximal toa genic region within a soybean genome, and exemplifying evidence ofrecombination further comprises a DNA of interest inserted into saidnongenic sequence. In accordance with embodiment 2, the recombinantsequence of embodiment 1 has the following characterstics:

a. the level of methylation of said nongenic sequence is 1% or less

b. said nongenic sequence shares less than 40% sequence identity withany other sequence contained in the soybean genome;

c. said nongenic sequence is located within a 40 Kb region of a known orpredicted expressive soybean coding sequence; and

d. said nongenic sequence exhibits a recombination frequency within thesoybean genome of greater than 0.01574 cM/Mb. In accordance withembodiment 3 the recombinant sequence of embodiment 1 or 2 is providedwherein said nongenic sequence comprises a maximum length of 5.73 Kb. Inaccordance with embodiment 4 the recombinant sequence of any ofembodiments 1-3 is provided, wherein said nongenic sequence comprises 1%or less nucleotide methylation. In accordance with embodiment 5 therecombinant sequence of any of embodiments 1-4 is provided, wherein saidnongenic sequence is 1 Kb to 5.73 Kb in length and contains nomethylated cytosine residues. In accordance with embodiment 6 therecombinant sequence of any of embodiments 1-5 is provided, wherein saidnongenic sequence does not align with greater than 40% sequence identityto any other sequence within the soybean genome. In accordance withembodiment 7 the recombinant sequence of any of claims 1-6 is provided,wherein said nongenic sequence exemplifies evidence of recombination ata recombination frequency of greater than 0.01574 cM/Mb. In accordancewith embodiment 8 the recombinant sequence of any of claims 1-7 isprovided, wherein a 40 Kb region of native soybean genome comprisingsaid nongenic sequence also comprises at least one known or predictedsoybean coding sequence, or a sequence comprising a 2 Kb upstream and/or1 Kb downstream sequence of a known soybean gene. In accordance withembodiment 9 the recombinant sequence of any of claims 1-8 is provided,wherein said known or predicted soybean coding sequence expresses asoybean protein. In accordance with embodiment 10 the recombinantsequence of any of claims 1-9 is provided, wherein said nongenicsequence does not contain a methylated polynucleotide. In accordancewith embodiment 11 the recombinant sequence of any of claims 1-10 isprovided, wherein one end of said nongenic sequence is within 40 Kb ofan expressed endogenous gene. In accordance with embodiment 12 therecombinant sequence of any of claims 1-11 is provided, wherein said DNAof interest comprises an analytical domain. In accordance withembodiment 13 the recombinant sequence of any of claims 1-12 isprovided, wherein said DNA of interest does not encode a peptide. Inaccordance with embodiment 14 the recombinant sequence of any of claims1-12 is provided, wherein said DNA of interest encodes a peptide,optionally encoding an insecticidal resistance gene, herbicide tolerancegene, nitrogen use efficiency gene, water use efficiency gene,nutritional quality gene, DNA binding gene, or selectable marker gene.In accordance with embodiment 16 the recombinant sequence of any ofclaims 1-14 is provided, wherein said recombinant sequence comprises thefollowing characteristics:

a. said nongenic sequence contains less than 1% DNA methylation

b. said nongenic sequence exhibits a 0.01574 to 83.52 cM/Mbrecombination frequency within the soybean genome;

c. said nongenic sequence exhibits a 0 to 0.494 level of nucleosomeoccupancy of the soybean genome;

d. said nongenic sequence shares less than 40% sequence identity withany other sequence contained in the soybean genome;

e. said nongenic sequence has a relative location value from 0 to0.99682 ratio of genomic distance from a soybean chromosomal centromere;

f. said nongenic sequence has a guanine/cytosine percent content rangeof 14.36 to 45.9%;

g. said nongenic sequence is located proximally to a genic sequence;and,

h. said nongenic sequence is located in a 1 Mb region of soybean genomicsequence that comprises one or more additional nongenic sequences.

In accordance with embodiment 17a soybean plant, soybean plant part, orsoybean plant cell comprising a recombinant sequence of any one ofembodiments 1-14 and 16. In accordance with embodiment 18 the soybeanplant, soybean plant part, or soybean plant cell of embodiment 17 isprovided, wherein said known or predicted soybean coding sequenceexpresses at a level ranging from 0.000415 to 872.7198. In accordancewith embodiment 19 the recombinant sequence of any of claim 1-14, 16 or17 is provided, wherein said DNA of interest and/or said nongenicsequence are modified during insertion of said DNA of interest into saidnongenic sequence.

In accordance with embodiment 20a method of making a transgenic plantcell comprising a DNA of interest targeted to one nongenic soybeangenomic sequenceis provided wherein the method comprises:

a. selecting an optimal nongenic soybean genomic locus;

b. introducing a site specific nuclease into a plant cell, wherein thesite specific nuclease cleaves said nongenic soybean genomic sequence;

c. introducing the DNA of interest into the plant cell;

d. targeting the DNA of interest into said nongenic locus, wherein thecleavage of said nongenic sequence facilitates integration of thepolynucleotide sequence into said nongenic locus; and

e. selecting transgenic plant cells comprising the DNA of interesttargeted to said nongenic locus.

EXAMPLES Example 1: Identification of Targetable Genomic Loci in Soybean

The soybean genome was screened with a bioinformatics approach usingspecific criteria to select optimal genomic loci for targeting of apolynucleotide donor. The specific criteria used for selecting thegenomic loci were developed using considerations for optimal expressionof a transgene within the plant genome, considerations for optimalbinding of genomic DNA by a site specific DNA-binding protein, andtransgenic plant product development requirements. In order to identifyand select the genomic loci, genomic and epigenomic datasets of thesoybean genome were scanned using a bioinformatics approach. Screeninggenomic and epigenomic datasets resulted in select loci which met thefollowing criteria: 1) hypomethylated and greater than 1 Kb in length;2) targetable via site specific nuclease-mediated integration of apolynucleotide donor; 3) agronomically neutral or non-genic; 4) regionsfrom which an integrated transgene can be expressed; and 5) regions withrecombination within/around the locus. Accordingly, a total of 7,018genomic loci (SEQ ID NO:1-SEQ ID NO:7,018) were identified using thesespecific criteria. The specific criteria are further described in detailbelow.

Hypomethylation

The soybean genome was scanned to select optimal genomic loci largerthan 1 Kb that were DNA hypomethylated. DNA methylation profiles of rootand shoot tissues isolated from Glycine Max cultivar Williams82 wereconstructed using a high throughput whole genome sequencing approach.Extracted DNA was subjected to bisulphite treatment that convertsunmethylated cytosines to uracils, but does not affect methylatedcytosines, and then sequenced using Illumina HiSeq technology (Krueger,F. et al. DNA methylome analysis using short bisulfite sequencing data.Nature Methods 9, 145-151 (2012)). The raw sequencing reads werecollected and mapped to the soybean c.v. Williams82 reference genomeusing the Bismark™ mapping software as described in Krueger F, AndrewsSR (2011) Bismark: a flexible aligner and methylation caller forBisulfite-Seq applications. Bioinformatics 27: 1571-1572). Since, duringthe bisulphite conversion process, cytosines in the DNA sequence thatare methylated do not get converted to uracils, occurence of cytosinebases in the sequencing data indicate the presence of DNA methylation.The reads that are mapped to the reference sequence were analyzed toidentify genomic positions of cytosine residues with support for DNAmethylation. The methylation level for each cytosine base in the genomewas calculated as a percentage of the number of methylated reads mappinga particular cytosine base location to the total number of reads mappingto that location. The following hypothetical explains how methylationlevels were calculated for each base within the soybean genome. Forexample, consider that there is a cytosine base at position 100 inchromosome 1 of the soybean c.v. Williams82 reference sequence. If thereare a total of 20 reads mapped to cytosine base at position 100, and 10of these reads are methylated, then the methylation level for thecytosine base at position 100 in chromosome 1 is estimated to be 50%.Accordingly, a profile of the methylation level for all of the genomicDNA base pairs obtained from the root and shoot tissue of soybean wascalculated. The reads that could not be correctly mapped to uniquelocations in the soybean genome matched repetitive sequences that arewidespread in the soybean genome, and are known in the art to bepredominantly methylated.

Using the above described protocol, the methylation levels for thesoybean c.v. Williams82 genome were measured. As such, regions of thesoybean genome containing methylated reads indicated that these regionsof the soybean genome were methylated. Conversely, the regions of thesoybean genome that were absent of methylated reads indicated theseregions of the soybean genome were non-methylated. The regions of thesoybean genome from the shoot and root tissues that were non-methylatedand did not contain any methylated reads are considered as“hypomethylated” regions. To make the root and shoot methylationprofiles available for visualization, wiggle plots(http://useast.ensembl.org/info/website/upload/wig.html) were generatedfor each of the soybean c.v. Williams82 chromosomes.

After obtaining the DNA methylation level at the resolution of a singlebase pair in root and shoot tissues, as described above, the soybeangenome was screened using 100 bp windows to identify genomic regionsthat are methylated. For each window screened in the genome, a DNAmethylation level was obtained by calculating the average level ofmethylation at every cytosine base in that window. Genomic windows witha DNA methylation level greater than 1% were termed as genomic regionsthat were methylated. The methylated windows identified in root andshoot profiles were combined to create a consensus methylation profile.Conversely, regions in the genome that did not meet these criteria andwere not identified as methylated regions in the consensus profile weretermed as hypo-methylated regions. Table 1 summarizes the identifiedhypo-methylated regions.

TABLE 1 Hypomethylation profile of soybean c.v. Williams82 genome. Totalsoybean c.v. Williams82 genome size ~970 Mb Total combined length ofhypomethylated region ~354 Mb (36.5% of the soybean c.v. Williams82genome) Number of hypomethylated regions above 100 Bp 763,709 Number ofhypomethylated regions above 1 Kb 94,745 Number of hypomethylatedregions above 2 Kb 19,369 Number of hypomethylated regions above 10 Kb354 Minimum length of hypomethylated region 100 Bp Maximum length ofhypomethylated region 84,100 Bp

These hypomethylated regions of the soybean c.v. WILLIAMS82 genome werefurther characterized to identify and select specific genomic loci asthe methylation free context of these regions indicated the presence ofopen chromatin. As such, all subsequent analyses were conducted on theidentified hypomethylated regions.

Targetability

The hypomethylated sites identified in the soybean c.v. WILLIAMS82 werefurther analyzed to determine which sites were targetable via sitespecific nuclease-mediated integration of a polynucleotide donor.Glycine max is known to be a paleopolyploid crop which has undergonegenome duplications in its genomic history (Jackson et al Genomesequence of the palaeopolyploid soybean, Nature 463, 178-183 (2010)).The soybean genome is known in the art to contain long stretches ofhighly repetitive DNA that are methylated and have high levels ofsequence duplication. Annotation information of known repetitive regionsin the soybean genome was collected from the Soybean Genome Database(www.soybase.org, Shoemaker R. C. et al. SoyBase the USDA-ARS so beangenetics and genomics database, Nucleic Acids Res. 2010 January: 38Database issue:D843-6.).

Accordingly, the hypomethylated sites identified above were screened toremove any sites that aligned with known repetitive regions annotated onthe soybean genome. The remaining hypomethylated sites that passed thisfirst screen were subsequently scanned using a BLAST™ based homologysearch of a soybean genomic database via the NCBI BLAST™+ software(version 2.2.25) run using default parameter settings (Stephen F.Altschul et al (1997) Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs. Nucleic Acids Res. 25:3389-3402). As aresult of the BLAST™ screen, any hypomethylated sites that hadsignificant matches elsewhere in the genome, with sequence alignmentcoverage of over 40%, were removed from further analyses.

Agronomically Neutral or Nongenic

The hypomethylated sites identified in the soybean c.v. William82 werefurther analyzed to determine which sites were agronomically neutral ornongenic. As such, the hypomethylated sites described above werescreened to remove any sites that overlapped or contained any known orpredicted endogenous soybean c.v. William82 coding sequences. For thispurpose, annotation data of known genes and mapping information ofexpressed sequence tag (EST) data were collected from Soybean GenomicDatabase (www.soybase.org—version 1.1 gene models were used, Jackson etal Genome sequence of the palaeopolyploid soybean Nature 463, 178-183(2010)). Any genomic region immediately 2 Kb upstream and 1 Kbdownstream to an open reading frame were also considered. These upstreamand downstream regions may contain known or unknown conserved regulatoryelements that are essential for gene function. The hypomethylated sitespreviously described above were analyzed for the presence of the knowngenes (including the 2 Kb upstream and 1 Kb downstream regions) andESTs. Any hypomethylated sites that aligned with or overlapped withknown genes (including the 2 Kb upstream and 1 Kb downstream regions) orESTs were removed from downstream analysis.

Expression

The hypomethylated sites identified in the soybean c.v. Williams82 werefurther analyzed to determine which sites were within proximity to anexpressed soybean gene. The transcript level expression of soybean geneswas measured by analyzing transcriptome profiling data generated fromsoybean c.v. Williams82 root and shoot tissues using RNAseq™ technologyas described in Mortazavi et al., Mapping and quantifying mammaliantranscriptomes by RNA-Seq. Nat. Methods. 2008; 5(7):621-628, andShoemaker R C et al., RNA-Seq Atlas of Glycine max: a guide to thesoybean Transcriptome. BMC Plant Biol. 2010 Aug. 5; 10:160. For eachhypomethylated site, an analysis was completed to identify any annotatedgenes present within a 40 Kb region in proximity of the hypomethylatedsite, and an average expression level of the annotated gene(s) locatedin proximity to the hypomethylated site. Hypomethylated sites locatedgreater than 40 Kb from an annotated gene with a non-zero averageexpression level were determined to not be proximal to an expressedsoybean gene and were removed from further analyses.

Recombination

The hypomethylated sites identified in the soybean c.v. Williams82 werefurther analyzed to determine which sites had evidence of recombinationand could facilitate introgression of the optimal genomic loci intoother lines of soybean via conventional breeding. Diverse soybeangenotypes are routinely crossed during conventional breeding to developnew and improved soybean lines containing traits of agronomic interest.As such, agronomic traits that are introgressed into optimal genomicloci within a soybean line via plant-mediated transformation of atransgene should be capable of further being introgressed into othersoybean lines, especially elite lines, via meiotic recombination duringconventional plant breeding. The hypomethylated sites described abovewere screened to identify and select sites that possessed some level ofmeiotic recombination. Any hypomethylated sites that were present withinchromosomal regions characterized as recombination “cold-spots” wereidentified and removed. In soybean, these cold spots were defined usinga marker dataset generated from recombinant inbred mapping population(Williams 82×P1479752). This dataset consisted of ˜16,600 SNP markersthat could be physically mapped to the Glycine max reference genomesequence.

The meiotic recombination frequencies between any pair of soybeangenomic markers across a chromosome were calculated based on the ratioof the genetic distance between markers (in centimorgan (cM)) to thephysical distance between the markers (in megabases (Mb)). For example,if the genetic distance between a pair of markers was 1 cM, and thephysical distance between the same pair of markers was 2 Mb, then thecalculated recombination frequency was determined to be 0.5 cM/Mb. Foreach hypomethylated site identified above, a pair of markers at least 1Mb apart was chosen and the recombination frequency was calculated.Deployment of this method was used to calculate the recombinationfrequency of the hypomethylated sites. Any hypomethylated sites with arecombination frequency of 0 cM/Mb were identified and removed fromfurther analysis. The remaining hypomethylated regions comprising arecombination frequency greater than 0 cM/Mb were selected for furtheranalysis.

Identification of Optimal Genomic Loci

Application of the selection criteria described above resulted in theidentification of a total of 90,325 optimal genomic loci from thesoybean genome. Table 2 summarizes the lengths of the identified optimalgenomic loci. These optimal genomic loci possess the followingcharacteristics: 1) hypomethylated genomic loci greater than 1 Kb inlength; 2) genomic loci that are targetable via site specificnuclease-mediated integration of a polynucleotide donor; 3) genomic locithat are agronomically neutral or nongenic; 4) genomic loci from which atransgene can be expressed; and 5) evidence of recombination within thegenomic loci. Of all of the optimal genomic loci described in Table 2,only the optimal genomic loci that were greater than 1 Kb were furtheranalyzed and utilized for targeting of a donor polynucleotide sequence.The sequences of these optiml genomic loci are disclosed as SEQ IDNO:1-SEQ ID NO:7,018. Collectively, these optimal genomic loci arelocations within the soybean genome that can be targeted with a donorpolynucleotide sequence, as further demonstrated herein below.

TABLE 2 Lists the size range of optimal genomic loci identified in thesoybean genome that are hypomethylated, show evidence of recombination,targetable, agronomically neutral or nongenic, and are in proximity toan expressed endogenous gene. Number of optimal genomic loci larger than100 Bp 90,325 Number of optimal genomic loci larger than 1 Kb 7,018Number of optimal genomic loci larger than 2 Kb 604 Number of optimalgenomic loci larger than 4 Kb 9

Example 2: F-Distribution and Principal Component Analysis to ClusterOptimal Genomic Loci from Soybean

The 7,018 identified optimal genomic loci (SEQ ID NO: 1-SEQ ID NO:7,018) were further analyzed using the F-distribution and PrincipalComponent Analysis statistical methods to define a representativepopulation and clusters for grouping of the optimal genomic loci.

F-Distribution Analysis

The identified 7,018 optimal genomic loci were statistically analyzedusing a continuous probability distribution statistical analysis. As anembodiment of the continuous probability distribution statisticalanalysis, an F-distribution test was completed to determine arepresentative number of optimal genomic loci. The F-distribution testanalysis was completed using equations and methods known by those withskill in the art. For more guidance, the F-distribution test analysis asdescribed in K. M Remund, D. Dixon, D L. Wright and L R. Holden.Statistical considerations in seed purity testing for transgenic traits.Seed Science Research (2001) 11, 101-119, herein incorporated byreference, is a non-limiting example of an F-distribution test. TheF-distribution test assumes random sampling of the optimal genomic loci,so that any non-valid loci are evenly distributed across the 7,018optimal genomic loci, and that the number of optimal genomic locisampled is 10% or less of the total population of 7,018 optimal genomicloci.

The F-distribution analysis indicated that 32 of the 7,018 optimalgenomic loci provided a representative number of the 7,018 optimalgenomic loci, at a 95% confidence level. Accordingly, the F-distributionanalysis showed that if 32 optimal genomic loci were tested and all weretargetable with a donor polynucleotide sequence, then these resultswould indicate that 91 or more of the 7,018 optimal genomic loci arepositive at the 95% confidence level. The best estimate of validatingthe total percentage of the 7,018 optimal genomic loci would be if 100%of the 32 tested optimal genomic loci were targetable. Accordingly, 91%is actually the lower bound of the true percent validated at the 95%confidence level. This lower bound is based on the 0.95 quantile of theF-distribution, for the 95% confidence level (Remund K, Dixon D, WrightD, and Holden L. Statistical considerations in seed purity testing fortransgenic traits. Seed Science Research (2001) 11, 101-119).

Principal Component Analysis

Next, a Principal Component Analysis (PCA) statistical method wascompleted to further assess and visualize similarities and differencesof the data set comprising the 7,018 identified optimal genomic loci toenable sampling of diverse loci for targeting validation. The PCAinvolves a mathematical algorithm that transforms a larger number ofcorrelated variables into a smaller number of uncorrelated variablescalled principal components.

The PCA was completed on the 7,018 identified optimal genomic loci bygenerating a set of calculable features or attributes that could be usedto describe the 7,018 identified optimal genomic loci. Each feature isnumerically calculable and is defined specifically to capture thegenomic and epigenomic context of the 7,018 identified optimal genomicloci. A set of 10 features for each soybean optimal genomic loci wasidentified and are described in greater detail below.

1. Length of the optimal genomic loci

-   -   a. The length of the optimal genomic loci in this data set        ranged from a minimum of 1,000 Bp to a maximum of 5,713 Bp.

2. Recombination frequency in a 1 MB region around the optimal genomicloci

-   -   a. In soybean, recombination frequency for a chromosomal        location was defined using an internal high resolution marker        dataset generated from multiple mapping populations.    -   b. Recombination frequencies between any pairs of markers across        the chromosome were calculated based on the ratio of the genetic        distance between markers (in centimorgan (cM)) to the physical        distance between the markers (in Mb). For example, if the        genetic distance between a pair of markers is 1 cM and the        physical distance between the same pairs of markers is 2 Mb, the        calculated recombination frequency is 0.5 cM/Mb. For each        optimal genomic loci, a pair of markers at least 1 Mb apart was        chosen and the recombination frequency was calculated in this        manner. These recombination values ranged from a minimum of        0.01574 cM/Mb to a maximum of 83.52 cM/Mb.

3. Level of optimal genomic loci sequence uniqueness

-   -   a. For each optimal genomic loci, the nucleotide sequence of the        optimal genomic loci was scanned against the soybean c.v.        Williams82 genome using a BLAST™ based homology search using the        NCBI BLAST™+ software (version 2.2.25) run using the default        parameter settings (Stephen F. Altschul et al (1997), “Gapped        BLAST and PSI-BLAST: a new generation of protein database search        programs”, Nucleic Acids Res. 25:3389-3402). As these optimal        genomic loci sequences are identified from the soybean c.v.        Williams82 genome, the first BLAST™ hit identified through this        search represents the soybean c.v. Williams82 sequence itself.        The second BLAST™ hit for each optimal genomic loci sequence was        identified and the alignment coverage (represented as the        percent of the optimal genomic loci covered by the BLAST™ hit)        of the hit was used as a measure of uniqueness of the optimal        genomic loci sequence within the soybean genome. These alignment        coverage values for the second BLAST™ hit ranged from a minimum        of 0% to a maximum of 39.97% sequence identity. Any sequences        that aligned at higher levels of sequence identity were not        considered.

4. Distance from the optimal genomic loci to the closest gene in itsneighborhood

-   -   a. Gene annotation information and the location of known genes        in the Soybean genome were extracted from Soybean Genome        Database (available at, www.soybase.org—version 1.1 gene models        were used, Jackson et al Genome sequence of the palaeopolyploid        soybean, Nature 463, 178-183 (2010)). For each optimal genomic        loci, the closest annotated gene, considering both upstream and        downstream locations, was identified and the distance between        the optimal genomic loci sequence and the gene was measured (in        Bp). For example, if a optimal genomic locus is located in        chromosome Gm01 from position 2,500 to position 3,500, and the        closest gene to this optimal genomic locus is located in        chromosome Gm01 from position 5,000 to position 6,000, the        distance from the optimal genomic loci to this closest gene is        calculated to be 1500 Bp. These values for all 7,018 of the        optimal genomic loci dataset ranged from a minimum of 1,001 Bp        to a maximum of 39,482 Bp.

5. GC % in the optimal genomic loci sequence

-   -   a. For each optimal genomic locus, the nucleotide sequence was        analyzed to estimate the number of Guanine and Cytosine bases        present. This count was represented as a percentage of the        sequence length of each optimal genomic locus and provides a        measure for GC %. These GC % values for the soybean optimal        genomic loci dataset range from 14.4% to 45.9%.

6. Number of genes in a 40 Kb neighborhood around the optimal genomicloci sequence

-   -   a. Gene annotation information and the location of known genes        in the soybean c.v. Williams82 genome were extracted from        Soybean Genome Database. For each of the 7,018 optimal genomic        loci sequence, a 40 Kb window around the optimal genomic loci        sequence was defined and the number of annotated genes with        locations overlapping this window was counted. These values        ranged from a minimum of 1 gene to a maximum of 18 genes within        the 40 Kb neighborhood.

7. Average gene expression in a 40 Kb neighborhood around the optimalgenomic loci

-   -   a. Transcript level expression of soybean genes was measured by        analyzing available transcriptome profiling data generated from        soybean c.v. Williams82 root and shoot tissues using RNAseq™        technology. Gene annotation information and the location of        known genes in the soybean c.v. Williams82 genome were extracted        from Soybean Genome Database For each optimal genomic locus,        annotated genes within the soybean c.v. Williams82 genome that        were present in a 40 Kb neighborhood around the optimal genomic        loci were identified. Expression levels for each of the genes        were extracted from the transcriptome profiles described in the        above referenced citations and an average gene expression level        was calculated. Expression values of all genes within the genome        of soybean vary greatly. The average expression values for all        of the 7,018 optimal genomic loci dataset ranged from a minimum        of 0.000415 to a maximum of 872.7198.

8. Level of nucleosome occupancy around the optimal genomic loci

-   -   a. Understanding the level of nucleosome occupancy for a        particular nucleotide sequence provides information about        chromosomal functions and the genomic context of the sequence.        The NuPoP™ statistical package was used to predict the        nucleosome occupancy and the most probable nucleosome        positioning map for any size of genomic sequences (Xi, L.,        Fondufe-Mittendor, Y., Xia, L., Flatow, J., Widom, J. and Wang,        J.-P., Predicting nucleosome positioning using a duration Hidden        Markov Model, BMC Bioinformatics, 2010,        doi:10.1186/1471-2105-11-346.). For each of the 7,018 optimal        genomic loci, the nucleotide sequence was submitted for analysis        with the NuPoP™ software and a nucleosome occupancy score was        calculated. These nucleosome occupancy scores for the soybean        optimal genomic loci dataset ranged from a minimum of 0 to a        maximum of 0.494.

9. Relative location within the chromosome (proximity to centromere)

-   -   a. A centromere is a region on a chromosome that joins two        sister chromatids. The portions of a chromosome on either side        of the centromere are known as chromosomal arms. Genomic        locations of centromeres on all 20 Soybean chromosomes were        identified in the published soybean c.v. Williams82 reference        sequence (Jackson et al Genome sequence of the palaeopolyploid        soybean Nature 463, 178-183 (2010)). Information on the position        of the centromere in each of the Soybean chromosomes and the        lengths of the chromosome arms was extracted from Soybean Genome        Database. For each optimal genomic locus, the genomic distance        from the optimal genomic locus sequence to the centromere of the        chromosome that it is located on, is measured (in Bp). The        relative location of optimal genomic loci within the chromosome        is represented as the ratio of its genomic distance to the        centromere relative to the length of the specific chromosomal        arm that it lies on. These relative location values for the        soybean optimal genomic loci dataset ranged from a minimum of 0        to a maximum of 0.99682 ratio of genomic distance.

10. Number of optimal genomic loci in a 1 Mb region

-   -   a. For each optimal genomic loci, a 1 Mb genomic window around        the optimal genomic loci location was defined and the number of        other, additional optimal genomic loci present within or        overlapping this region were calculated, including the optimal        genomic loci under consideration. The number of optimal genomic        loci in a 1 Mb ranged from a minimum of 1 to a maximum of 49.

All of the 7,018 optimal genomic loci were analyzed using the featuresand attributes described above. The results or values for the score ofthe features and attributes of each optimal genomic locus are furtherdescribed in Table 3 (herein incorporated by reference as a separateelectronic filing). The resulting dataset was used in the PCAstatistical method to cluster the 7,018 identified optimal genomic lociinto clusters. During the clustering process, after estimating the “p”principle components of the optimal genomic loci, the assignment of theoptimal genomic loci to one of the 32 clusters proceeded in the “p”dimensional Euclidean space. Each of the “p” axes was divided into “k”intervals. Optimal genomic loci assigned to the same interval weregrouped together to form clusters. Using this analysis, each PCA axiswas divided into two intervals, which was chosen based on a prioriinformation regarding the number of clusters required for experimentalvalidation. All analysis and the visualization of the resulting clusterswere carried out with the Molecular Operating Environment™ (MOE)software from Chemical Computing Group Inc. (Montreal, Quebec, Canada).

The PCA approach was used to cluster the set of 7,018 identified optimalgenomic loci into 32 distinct clusters based on their feature values,described above. During the PCA process, five principal components (PC)were generated, with the top three PCs containing about 90% of the totalvariation in the dataset (Table 4). These three PCAs were used tographically represent the 32 clusters in a three dimensional plot (FIG.1). After the clustering process, was completed, one representativeoptimal genomic locus was chosen from each cluster. This was performedby choosing a select optimal genomic locus, within each cluster, thatwas closest to the centroid of that cluster (Table 4). The chromosomallocations of the 32 representative optimal genomic loci are uniformlydistributed among the 20 soybean chromosomes and are not biased towardany particular genomic location, as shown in FIG. 2.

TABLE 4 Description of the 32 soybean representative optimal genomicloci identified from the PCA Optimal Genomic Loci Length Cluster SEQ IDName Genomic Location (Bp) Number NO: soy_ogl_2474 Gm08: 2764201 . . .2766752 2552 1 1 soy_ogl_768 Gm03: 339101 . . . 341100 2000 2 506soy_ogl_2063 Gm06: 43091928 . . . 43094600 2673 3 748 soy_ogl_1906 Gm06:11576991 . . . 11578665 1675 4 1029 soy_ogl_1112 Gm03: 46211408 . . .46213400 1993 5 1166 soy_ogl_3574 Gm10: 46279901 . . . 46281026 1126 61452 soy_ogl_2581 Gm08: 9631801 . . . 9632800 1000 7 1662 soy_ogl_3481Gm10: 40763663 . . . 40764800 1138 8 1869 soy_ogl_1016 Gm03: 41506001 .. . 41507735 1735 9 2071 soy_ogl_937 Gm03: 37707001 . . . 37708600 160010 2481 soy_ogl_6684 Gm20: 1754801 . . . 1755800 1000 11 2614soy_ogl_6801 Gm20: 36923690 . . . 36924900 1211 12 2874 soy_ogl_6636Gm19: 49977101 . . . 49978357 1257 13 2970 soy_ogl_4665 Gm14: 5050547 .. . 5051556 1010 14 3508 soy_ogl_3399 Gm10: 6612501 . . . 6613500 100015 3676 soy_ogl_4222 Gm13: 23474923 . . . 23476100 1178 16 3993soy_ogl_2543 Gm08: 7532001 . . . 7534800 2800 17 4050 soy_ogl_275 Gm01:51869201 . . . 51870400 1200 18 4106 soy_ogl_598 Gm02: 41665601 . . .41667900 2300 19 4496 soy_ogl_1894 Gm06: 10540801 . . . 10542300 1500 204622 soy_ogl_5454 Gm17: 1944101 . . . 1945800 1700 21 4875 soy_ogl_6838Gm20: 38263922 . . . 38265300 1379 22 4888 soy_ogl_4779 Gm14: 45446301 .. . 45447700 1400 23 5063 soy_ogl_3333 Gm10: 2950701 . . . 2951800 110024 5122 soy_ogl_2546 Gm08: 7765875 . . . 7767500 1626 25 5520soy_ogl_796 Gm03: 1725501 . . . 1726600 1100 26 5687 soy_ogl_873 Gm03:33650665 . . . 33653000 2336 27 6087 soy_ogl_5475 Gm17: 3403108 . . .3404200 1093 28 6321 soy_ogl_2115 Gm07: 1389701 . . . 1390900 1200 296520 soy_ogl_2518 Gm08: 5229501 . . . 5230667 1167 30 6574 soy_ogl_5551Gm17: 6541901 . . . 6543200 1300 31 6775 soy_ogl_4563 Gm13: 38977701 . .. 38978772 1072 32 6859Final Selection of Genomic Loci for Targeting of a Polynucleotide DonorPolynucleotide Sequence

A total of 32 genomic loci were identified and selected for targetingwith a donor polynucleotide sequence from the 7,018 genomic loci thatwere clustered within 32 distinct clusters. For each of the 32 clusters,a representative genomic locus (closest to the centroid of the clusteras described above in Table 4) or an additional locus with homology totargeting line were chosen. The additional optimal genomic loci wereselected by first screening all of the 7,018 selected optimal genomicsequences against a whole genome database consisting of genomic DNAsequence data for both Glycine max c.v. Maverick (transformation andtargeting screening line) and Glycine max c.v. Williams82 (referenceline) to determine the coverage (how many optimal genomic loci werepresent in both genomes) and percentage of sequence identity in thegenome from both lines. The optimal genomic loci with 100% coverage (theentire sequence length of the optimal loci aligned between both genomes)and 100% identity in the Williams82 genomic databases were selected fortargeting validation. Other criteria such as genomic loci size, extentof uniqueness, GC % content and chromosomal distribution of the optimalgenomic loci were also taken into consideration in selecting theadditional optimal genomic loci. The chromosomal location of the 32selected optimal genomic loci and the specific genomic configuration ofeach soybean optimal genomic loci are shown in FIG. 3 and Table 5,respectively.

TABLE 5 Table 5. Description of the 32 soybean selected optimal genomicloci chosen for targeting validation. From these optimal genomic locilisted in this table, exemplification of cleavage and targeting of 32soybean optimal genomic loci are representative of the identified totalof 7,018 soybean selected optimal genomic loci. Optimal Genomic LengthCluster SEQ ID Loci Name Genomic Location (Bp) Number NO: soy_ogl_308Gm02: 1204801 . . . 1209237 4437 1 43 soy_ogl_307 Gm02: 1164701 . . .1168400 3700 2 566 soy_ogl_2063 Gm06: 43091928 . . . 43094600 2673 3 748soy_ogl_1906 Gm06: 11576991 . . . 11578665 1675 4 1029 soy_ogl_262 Gm01:51061272 . . . 51062909 1638 5 1376 soy_ogl_5227 Gm16: 1298889 . . .1300700 1812 6 1461 soy_ogl_4074 Gm12: 33610401 . . . 33611483 1083 71867 soy_ogl_3481 Gm10: 40763663 . . . 40764800 1138 8 1869 soy_ogl_1016Gm03: 41506001 . . . 41507735 1735 9 2071 soy_ogl_937 Gm03: 37707001 . .. 37708600 1600 10 2481 soy_ogl_5109 Gm15: 42391349 . . . 42393400 205211 2639 soy_ogl_6801 Gm20: 36923690 . . . 36924900 1211 12 2874soy_ogl_6636 Gm19: 49977101 . . . 49978357 1257 13 2970 soy_ogl_4665Gm14: 5050547 . . . 5051556 1010 14 3508 soy_ogl_6189 Gm18: 55694401 . .. 55695900 1500 15 3682 soy_ogl_4222 Gm13: 23474923 . . . 23476100 117816 3993 soy_ogl_2543 Gm08: 7532001 . . . 7534800 2800 17 4050soy_ogl_310 Gm02: 1220301 . . . 1222300 2000 18 4326 soy_ogl_2353 Gm07:17194522 . . . 17196553 2032 19 4593 soy_ogl_1894 Gm06: 10540801 . . .10542300 1500 20 4622 soy_ogl_3669 Gm11: 624301 . . . 626200 1900 214879 soy_ogl_3218 Gm09: 40167479 . . . 40168800 1322 22 4932soy_ogl_5689 Gm17: 15291601 . . . 15293400 1800 23 5102 soy_ogl_3333Gm10: 2950701 . . . 2951800 1100 24 5122 soy_ogl_2546 Gm08: 7765875 . .. 7767500 1626 25 5520 soy_ogl_1208 Gm04: 4023654 . . . 4025650 1997 265698 soy_ogl_873 Gm03: 33650665 . . . 33653000 2336 27 6087 soy_ogl_5957Gm18: 6057701 . . . 6059100 1400 28 6515 soy_ogl_4846 Gm15: 924901 . . .926200 1300 29 6571 soy_ogl_3818 Gm11: 10146701 . . . 10148200 1500 306586 soy_ogl_5551 Gm17: 6541901 . . . 6543200 1300 31 6775 soy_ogl_7Gm05: 32631801 . . . 32633200 1400 32 6935 soy_OGL_684 Gm02: 45903201 .. . 45907300 4100 1 47 soy_OGL_682 Gm02: 45816543 . . . 45818777 2235 92101 soy_OGL_685 Gm02: 45910501 . . . 45913200 2700 1 48 soy_OGL_1423Gm04: 45820631 . . . 45822916 2286 2 639 soy_OGL_1434 Gm04: 46095801 . .. 46097968 2168 1 137 soy_OGL_4625 Gm14: 3816738 . . . 3820070 3333 1 76soy_OGL_6362 Gm19: 5311001 . . . 5315000 4000 1 440

A large suite of 7,018 genomic locations have been identified in thesoybean genome as optimal genomic loci for targeting with a donorpolynucleotide sequence using precision genome engineering technologies.A statistical analysis approach was deployed to group the 7,018 selectedgenomic loci into 32 clusters with similar genomic contexts, and toidentify a subset of 32 selected genomic loci representative of the setof 7,018 selected genomic loci. The 32 representative loci werevalidated as optimal genomic loci via targeting with a donorpolynucleotide sequence. By performing the PCA statistical analysis forthe numerical values generated for the ten sets of features orattributes that are described above, the ten features or attributes werecomputed into PCA components of fewer dimensions. As such, PCAcomponents were reduced into five dimensions that are representative ofthe ten features or attributes described above (Table 6). Each PCAcomponent is equivalent to a combination of the ten features orattributes described above. From these PCA components comprising fivedimensions, as computed using the PCA statistical analysis, the 32clusters were determined.

TABLE 6 The five PCA components (PCA1, PCA2, PCA3, PCA4, and PCA5) thatdefine each of the 32 clusters and the sequences (SEQ ID NO: 1-SEQ IDNO: 7,018) which make up each cluster. These five dimensions arerepresentative of the ten features or attributes described above thatwere used to identify the optimal genomic loci. The minimum (Min), mean,median and maximum (Max) values for each PCA component are provided.Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5 Cluster 6 Cluster 7Cluster 8 Cluster 9 Cluster 10 Cluster 11 (SEQ ID (SEQ ID (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 1-- NO:506-- NO: 748-- NO: 1029-- NO: 1166-- NO: 1452-- NO: 1662-- NO: 1869--NO: 2071-- NO: 2481-- NO: 2614-- SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 505) NO: 747) NO: 1028) NO:1165) NO: 1451) NO: 1661) NO: 1868) NO: 2070) NO: 2480) NO: 2613) NO:2873) PCA Min −1.70777 0.022046 −4.54911 −1.72266 −0.36976 0.287697−3.34863 −1.0806 −1.5084417 −0.06921 −4.85854 1 Mean 0.349775 0.812634−1.47305 −0.00185 0.540899 0.967917 −0.58528 0.313491 0.1781458250.746656 −1.77485 Median 0.363103 0.796321 −1.18164 0.049082 0.524980.918269 −0.34364 0.291582 0.204892845 0.729936 −1.59613 Max 1.5078941.834871 0.032399 2.027233 1.499719 2.461219 0.417058 1.718384 1.44528232.258209 −0.10355 PCA Min −0.65485 −0.6907 −1.37642 −1.15246 −2.2623−2.69847 −2.33499 −2.05394 −0.85615188 −1.07918 −1.48917 2 Mean 0.8035910.805611 0.42863 0.549053 −0.97646 −0.63594 −1.07926 −0.67684 0.01310180.201017 −0.12584 Median 0.640172 0.690953 0.30208 0.435896 −0.92946−0.51848 −1.03176 −0.62625 −0.061526693 0.165577 −0.16842 Max 6.750384.21356 3.492035 2.037537 0.224862 0.316075 0.014994 0.262266 2.87375931.883538 2.389063 PCA Min −4.63386 −6.20928 −3.64977 −7.46971 −2.4347−3.28026 −2.79672 −2.36222 −1.7842444 −3.17428 −2.64864 3 Mean −1.0374−0.87017 −1.09511 −1.21149 −0.49711 −0.30392 −0.4893 −0.36718−0.137149779 −0.20772 −0.28997 Median −0.94654 −0.7282 −0.92816 −0.96309−0.45901 −0.19996 −0.43677 −0.27515 −0.068803158 −0.04455 −0.18716 Max0.240454 0.010148 −0.11534 −0.13414 0.476554 0.457804 0.452481 0.4535050.9092167 0.928412 0.782125 PCA Min −2.22011 −1.02405 −1.33923 0.069312−1.70627 −0.80904 −1.29231 0.360563 −2.9615474 −2.44418 −2.7613 4 Mean−0.71495 0.283541 0.212841 1.084988 −0.35855 0.479481 0.459736 1.348666−1.407512305 −0.75615 −0.85361 Median −0.70787 0.306108 0.2090551.116651 −0.35772 0.435449 0.436138 1.307628 −1.38790425 −0.78738−0.81593 Max 0.786678 1.575184 2.221794 2.571196 0.755949 2.6648172.193427 3.122114 −0.40942505 0.783523 0.985444 PCA Min −0.17971−3.06393 −0.53749 −4.5557 0.159064 −2.0539 −0.70289 −1.90857 −1.897981−4.47156 −2.35152 5 Mean 0.943093 0.368965 0.713771 −0.21905 0.8767450.463248 0.768677 0.285719 −0.029561107 −0.90424 −0.18625 Median0.854279 0.3771 0.670629 −0.10817 0.846543 0.459296 0.763885 0.3389910.034177913 −0.68409 −0.12264 Max 3.583402 2.613815 2.279238 2.3414781.913726 1.633977 2.164417 1.422805 0.84637429 0.242494 0.940019 Cluster12 Cluster 13 Cluster 14 Cluster 15 Cluster 16 Cluster 17 Cluster 18Cluster 19 Cluster 20 Cluster 21 Cluster 22 (SEQ ID (SEQ ID (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 2874--NO: 2970-- NO: 3508-- NO: 3676-- NO: 3993-- NO: 4050-- NO: 4106-- NO:4496-- NO: 4622-- NO: 4875-- NO: 4888-- SEQ ID SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 2969) NO: 3507) NO:3675) NO: 3992) NO: 4049) NO: 4105) NO: 4495) NO: 4621) NO: 4874) NO:4887) NO: 5062) PCA Min −2.10567 −0.78413 −0.1362 −3.50478 −1.06581−0.48995 −0.12394 −3.44417 −2.5926 0.041919 0.192262 1 Mean 0.2152540.402511 0.841125 −0.99405 0.054644 0.218477 0.705185 −1.60324 −0.219890.498017 0.919859 Median 0.167943 0.421486 0.793343 −0.86435 0.0433140.186449 0.69823 −1.62442 −0.07645 0.530588 0.860249 Max 2.6381221.521265 2.011089 0.254192 1.078006 1.212386 1.894809 −0.14778 1.105930.937608 1.90419 PCA Min −3.24885 −2.49287 −2.07915 −2.50642 −2.602890.060129 0.131404 −0.33368 −0.05632 −1.56352 −1.38005 2 Mean −0.53611−1.09247 −0.94959 −1.29395 −1.20352 1.419729 1.150768 1.001573 0.843814−0.4111 −0.07498 Median −0.33651 −1.08189 −0.91699 −1.24996 −1.176791.280417 1.065186 0.789798 0.776486 −0.28559 −0.02529 Max 2.608386−0.24001 0.020389 −0.4655 −0.31958 3.913198 3.040107 6.340514 2.9297410.123387 0.788801 PCA Min −14.6314 −1.01198 −1.91077 −1.7135 −2.73956−1.73844 −1.13076 −1.78506 −0.92532 0.00053 −0.17801 3 Mean −3.02840.137956 0.208329 0.071922 −0.21452 −0.27811 0.161876 −0.14195 0.2339530.437291 0.567525 Median −1.93463 1.177648 0.306399 0.132791 −0.00072−0.15148 0.163129 −0.06546 0.23185 0.450869 0.560588 Max 0.722841.034171 1.086972 0.996862 0.765974 0.427866 1.323874 0.948736 1.4092770.918483 1.635718 PCA Min −1.00771 −2.10637 −1.17239 −1.48955 −0.78727−1.60097 −0.69878 −1.09012 0.172103 −0.51316 −0.24073 4 Mean 0.594551−0.85746 −0.33529 −0.37717 0.438916 −0.23831 0.47476 0.670052 1.2102190.100213 0.936613 Median 0.392421 −0.86062 −0.4333 −0.47105 0.356632−0.21174 0.451745 0.638494 1.196036 0.075167 0.954165 Max 4.860240.27396 0.580863 0.978394 2.500934 0.871996 1.775638 2.468554 2.6142630.536589 2.449815 PCA Min −18.7726 −0.77506 −3.53913 −1.20206 −3.511250.008136 −0.77069 −0.62934 −1.42543 0.258308 −0.57322 5 Mean −4.219430.229577 −0.3992 0.08327 −0.93398 0.701934 0.233117 0.369827 −0.023770.648857 0.282176 Median −2.90093 0.240883 −0.33338 0.087451 −0.705130.602369 0.225125 0.29277 −0.01138 0.603284 0.263371 Max −0.334011.115681 0.396515 1.044241 0.040091 2.01268 1.665714 1.937356 1.7917941.079582 1.412132 Cluster 23 Cluster 24 Cluster 25 Cluster 26 Cluster 27Cluster 28 Cluster 29 Cluster 30 Cluster 31 Cluster 32 (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:5063−− NO: 5122−− NO: 5520−− NO: 5687−− NO: 6087−− NO: 6321−− NO: 6520−−NO: 6574−− NO: 6775−− NO: 6589−− SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID NO: 5121) NO: 5519) NO: 5686) NO: 6086)NO: 6320) NO: 6519) NO: 6573) NO: 6774) NO: 6588) NO: 7018) PCA1 Min−3.01097 −2.17545 −1.47203 −0.82652 −4.22215 −2.8128 −0.55955 −0.14823−3.68328 −2.1948 Mean −1.10273 0.189203 0.026625 0.609339 −1.7704−0.5307 0.365085 0.695365 −1.17291 −0.20762 Median −1.05343 1.2740060.000548 0.622131 −1.68559 −0.44093 0.372341 0.679272 −1.05591 −0.07481Max 0.215051 1.539568 1.204076 2.040596 −0.20599 1.026142 1.0827781.542552 −0.04543 1.044939 PCA2 Min −1.37504 −1.29996 −0.49733 −0.35268−0.8685 −0.39322 −1.70938 −1.71589 −1.7904 −1.39851 Mean −0.52564−0.24039 0.701345 0.996746 0.520887 0.744232 −0.66621 −0.41853 −0.82975−0.40126 Median −0.48207 −1.18651 0.497651 0.817435 0.384759 0.686377−0.69103 −0.33802 −0.8047 −0.39539 Max 0.176255 0.87503 3.8805864.311936 3.021218 3.474901 0.015191 0.704506 −0.11251 0.595757 PCA3 Min−0.26777 −0.37688 −0.16467 −0.23246 −0.47501 −0.00111 0.538379 0.4351680.337445 0.222405 Mean 0.293 0.505379 0.71742 1.077263 0.78187 1.0054291.055894 1.156804 1.045676 1.227425 Median 0.264634 0.473588 0.6747811.053549 0.727749 1.007761 1.085427 1.14325 1.036725 1.200613 Max0.932042 1.841691 2.234525 2.790854 2.556613 2.483974 1.565017 3.161821.973707 2.509435 PCA4 Min 0.213129 0.305131 −3.03078 −3.22656 −2.78298−0.8606 −1.61996 −1.16215 −1.17888 −0.50044 Mean 1.158602 1.628149−1.25321 −0.49661 −0.42577 0.564935 −0.67182 −0.02267 0.109856 0.973061Median 1.088302 1.633074 −1.21889 −0.46274 −0.40653 0.537203 −0.72684−0.03125 0.201603 0.930979 Max 2.046121 2.833294 −0.00705 1.3012371.30015 2.491648 0.122389 1.218455 1.682597 2.614142 PCA5 Min −0.00876−1.17026 −2.49874 −3.23886 −2.76649 −4.07782 −0.9589 −1.70771 −1.41474−2.02076 Mean 0.59606 0.172591 −0.46655 −0.94402 −0.73067 −1.02591−0.15265 −0.47755 −0.34837 −0.70651 Median 0.577423 0.151654 −0.39547−0.84381 −0.69992 −0.89159 −0.17176 −0.40794 −0.32927 −0.61785 Max1.322481 1.497953 0.404344 0.182532 0.480085 −0.10675 0.562286 0.4460260.347785 −0.0103

Example 3: Design of Zinc Fingers to Bind Genomic Loci in Soybean

Zinc finger proteins directed against the identified DNA sequences ofthe representative genomic loci were designed as previously described.See, e.g., Urnov et al., (2005) Nature 435:646-551. Exemplary targetsequence and recognition helices are shown in Table 7 (recognition helixregions designs) and Table 8 (target sites). In Table 8, nucleotides inthe target site that are contacted by the ZFP recognition helices areindicated in uppercase letters and non-contacted nucleotides areindicated in lowercase. Zinc Finger Nuclease (ZFN) target sites weredesigned for all of the previously described 32 selected optimal genomicloci. Numerous ZFP designs were developed and tested to identify thefingers which bound with the highest level of efficiency with 32different representative genomic loci target sites which were identifiedand selected in soybean as described above. The specific ZFP recognitionhelices (Table 7) which bound with the highest level of efficiency tothe zinc finger recognition sequences were used for targeting andintegration of a donor sequence within the soybean genome.

TABLE 7 zinc finger designs for the soybean selected genomic loci(N/A indicates ″not applicable″). pDAB ZFP Number Number F1 F2 F3 F4 F5F6 124201 391 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO:NO: NO: 7019 7020 7021 7022 7023 7024 QSANRTK HRSSLRR QSANRTK DSSDRKKDRSNRTT DNSNRIK SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO:NO: NO: NO: 7025 7026 7027 7028 7029 7030 RSDNLSV QKATRIN RSDHLSERNDNRKN DRSNRTT RKYYLAK 124221 411 SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDN/A NO: NO: NO: NO: NO: 7031 7032 7033 7034 7035 DRSNRTT QSAHRIT HAQGLRHQSGHLSR QSGHLSR SEQ ID SEQ ID SEQ ID SEQ ID N/A N/A NO: NO: NO: NO: 70367037 7038 7039 QSGSLTR RLDWLPM RPYTLRL DNSNRIK 125332 651 SEQ ID SEQ IDSEQ ID SEQ ID N/A N/A NO: NO: NO: NO: 7040 7041 7042 7043 TSGNLTRTSGNLTR QSGDLTR HKWVLRQ  SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO:NO: NO: NO: 7044 7045 7046 7047 7048 QSGHLAR TSSNRKT DSSDRKK QSGNLARHNSSLKD 125309 655 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO:NO: NO: NO: 7049 7050 7051 7052 7053 7054 TSGSLSR QLNNLKT QSADRTKDNSNRIK TSGSLSR QSGDLTR SEQ ID SEQ ID SEQ ID SEQ ID N/A N/A NO: NO: NO:NO: 7055 7056 7057 7058 QSANRTK DRSNRTT QSGDLTR HRSSLLN 124884 195SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7059 70607061 7062 7063 IDHGRYR DRSNLTR QSGDLTR QSGDLTR QRNARTL SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7064 7065 7066 70677068 7069 TSGNLTR DRTGLRS SQYTLRD TSGHLSR RSDHLSE QSASRKN 124234 424SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7070 70717072 7073 7074 TNQNRIT HSNARKT QSADRTK DNSNRIK RSDALTQ SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7075 7076 7077 70787079 7080 TSGNLTR QSNQLRQ QSGNLAR RQEHRVA QSGALAR QSGHLSR 124257 447SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 70817082 7083 7084 7085 7086 QSGSLTR WRSCRSA QSGNLAR WRISLAA QKHHLGD RSADLSRSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7087 70887089 7090 7091 DRSNRTT QSANRTK QSANRTK DRSNRTT QSGNLAR 125316 662 SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7092 70937094 7095 7096 7097 QSGNLAR TSGNLTR DRSNRTT QNATRIN TSSNRKT QSGHLSRSEQ ID SEQ ID SEQ ID SEQ ID N/A N/A NO: NO: NO: NO: 7098 7099 7100 7101DSSTRKT QSGNLAR RSDVLST QSGPLTQ 124265 455 SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID N/A NO: NO: NO: NO: NO: 7102 7103 7104 7105 7106 QSGNLAR DKSCLPTWELNRRT TSGNLTR DRSNLTR SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO:NO: NO: NO: NO: NO: 7107 7108 7109 7110 7111 7112 DRSDLSR RREHLRARSDNLAR QWNYRGS RSHSLLR RRDTLLD 124273 463 SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7113 7114 7115 7116 7117 7118QSGDLTR QSGNLAR HQCCLTS RSANLTR RSANLAR TNQNRIT SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7119 7120 7121 7122 71237124 ATKDLAA TSGHLSR RSDNLSE TSSNRKT DRSALAR RSDYLAK 124888 213 SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7125 71267127 7128 7129 7130 rsdnlar qsnalnr qkgtlge qsgsltr rsdsllr wccclrdSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7131 71327133 7134 7135 qsgsltr drsyrnt dqsnlra rhshlts qsgnlar 124885 215 SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7136 71377138 7139 7140 7141 tsgnltr lsqdlnr rsdslsr dssartk rsdhlsa crrnlrnSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 71427143 7144 7145 7146 7147 seadrsk drsnltr drsalsr tssnrkt ergtlar drsalar124610 480 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO:NO: 7148 7149 7150 7151 7152 7153 STDYRYP QSGNLAR RSDNLSV TRWWLPERSDHLSQ TRSPLTT SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO:NO: NO: NO: 7154 7155 7156 7157 7158 7159 TNQSLHW QSGNLAR RPYTLRLQSGSLTR RSDVLSE TSSNRKT 124614 484 SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDN/A NO: NO: NO: NO: NO: 7160 7161 7162 7163 7164 RSDVLST RNSYLIS RSAHLARTNQNRIT RSDNLSV SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO:NO: NO: NO: 7165 7166 7167 7168 7169 7170 RSDHLSA RSANLTR LRHHLTRDRSTLRQ HNHDLRN TSGNLTR 124636 506 SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDN/A NO: NO: NO: NO: NO: 7171 7172 7173 7174 7175 QSANRTT QHAHRKT QSGNLARQRNHRTT QSANRTK SEQ ID SEQ ID SEQ ID SEQ ID N/A N/A NO: NO: NO: NO: 71767177 7178 7179 RSDHLSE TSGSLTR QSGALAR QSGHLSR 124648 518 SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7180 7181 7182 71837184 7185 YRWLRNS TNSNRKR QSANRTT HRSSLRR RSDVLSA QNATRIN SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7186 7187 7188 71897190 7191 RSDSLLR QSCARNV RPYTLRL HRSSLRR RSDSLLR QSCARNV 121225 233SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 71927193 7194 7195 7196 7197 QSSDLSR YHWYLKK QSANRTK DNSNRIK QSGNLAR DRTNLNASEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 71987199 7200 7201 7202 7203 RSDNLSE TSANLSR QSANRTK DNSYLPR LKQNLDA RSHHLKA121227 235 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO:7204 7205 7206 7207 7208 RSDHLSQ TARLLKL RSDNLTR QSSDLSR YHWYLKK SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7209 72107211 7212 7213 7214 DRSNLSR TSGNLTR DRSNRTT TNSNRKR RSDSLSV QNANRKT121233 241 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO:7215 7216 7217 7218 7219 TSGNLTR QRSHLSD RSDNLSE VRRALSS RSDNLSV SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7220 72217222 7223 7224 7225 QSSNLAR TSGSLTR QSGNLAR QKVNRAG TSGSLSR DSSALAK121235 243 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO:NO: 7226 7227 7228 7229 7230 7231 QSGDLTR RKDPLKE QSGNLAR ATCCLAHQSSDLSR RRDNLHS SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEO ID NO: NO: NO:NO: NO: NO: 7232 7233 7234 7235 7236 7237 QSGNLAR HNSSLKD QSGALARQSANRTK RSDHLST RSDHLSR 121238 250 SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDN/A NO: NO: NO: NO: NO: 7238 7239 7240 7241 7242 TSGNLTR DSTNLRA DRSHLARRSDDLTR TSSNRKT SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO:NO: NO: NO: 7243 7244 7245 7246 7247 7248 TSGNLTR QSGALVI QNAHRKTLKHHLTD RSDNLST DRSNRKT 121246 259 SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDN/A NO: NO: NO: NO: NO: 7249 7250 7251 7252 7253 DRSALSR RSDALTQ DRSTRTKQSGNLHV RSDNLTR SEQ ID SEQ ID SEQ ID SEQ ID N/A N/A NO: NO: NO: NO: 72547255 7256 7257 DRSNLSR QSGNLAR RSDSLLR WLSSLSA 121249 262 SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7258 7259 7260 72617262 7263 RSDNLST DSSSRIK QSGALAR QSGNLHV RSDVLST RYAYLTS SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7264 7265 7266 7267 7268RSDNLSE TRSPLRN QNAHRKT RSDHLSE RNDNRKN 125324 670 SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7269 7270 7271 7272 7273 QRTNLVEASKTRTN RSANLAR RSDHLTQ RSAHLSR SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/ANO: NO: NO: NO: NO: 7274 7275 7276 7277 7278 RSDNLSV QNANRIT DQSNLRAQNAHRKT RSAHLSR 121265 282 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO:NO: NO: NO: NO: NO: 7279 7280 7281 7282 7283 7284 DRSALAR RSDYLAKRSDDLSR RNDNRTK RSDHLST HSNTRKN SEQ ID SEQ ID SEQ ID SEQ ID N/A N/A NO:NO: NO: NO: 7285 7286 7287 7288 RSDVLSE QRSNLKV QSSNLAR QSGHLSR 121271288 SEQ ID SEQ ID SEQ ID SEQ ID N/A N/A NO: NO: NO: NO: 7289 7290 72917292 DRSDLSR LRFNLRN RSDSLSV QNANRKT SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDN/A NO: NO: NO: NO: NO: 7293 7294 7293 7296 7297 QSGDLTR TSGSLTR RSDDLTRYRWLLRS QSGDLTR 124666 538 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO:NO: NO: NO: NO: NO: 7298 7299 7300 7301 7302 7303 RSDNLST AACNRNARPYTLRL QSGSLTR SQYTLRD TSGHLSR SEQ ID SEQ ID SEQ ID SEQ ID N/A N/A NO:NO: NO: NO: 7304 7305 7306 7307 QSANRTK DRSNRTT RSDVLST CRRNLRN 124814598 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7308 73097310 7311 7312 QSGDLTR HRSSLLN TNQSLHW QSGNLAR QSGNLAR SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7313 7314 7315 73167317 7318 RSCCLHL RNASRTR QSGNLAR RQEHRVA RSDNLSE TSSNRKT 124690 560SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 73197320 7321 7322 7323 7324 RSDVLSE QRSNLKV QSGALAR YRWLRNS QSANRTT DRSNRTTSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 73257326 7327 7328 7329 7330 QNAHRKT LAHHLVQ HAQGLRH QSGHLSR RSDDLTR RRFTLSK124815 599 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO:NO: 7331 7332 7333 7334 7335 7336 RSDNLSE KSWSRYK RSAHLSR RSDDLTRYSWTLRD TSGNLTR SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO:NO: 7337 7338 7339 7340 7341 RSDVLST DNSSRTR RSDALAR RSDSLSA DRSDLSR124816 600 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO:7342 7343 7344 7345 7346 GTQGLGI DRSNLTR RNDDRKK RSDVLSE RSSDRTK SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7347 7348 7349 73507351 QSANRTK DSSHRTR QSANRTK SVGNLNQ TSGNLTR 124842 631 SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: 7352 7353 7354 73557356 7357 TNQNRIT HSNARKT QSSHLTR RLDNRTA QSGNLAR QGANLIK SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID N/A NO: NO: NO: NO: NO: 7358 7359 7360 7361 7362RSDHNLST QKSPLNT QSSDLSR QSSDLSR YHWYLKK 125338  37 SEQ ID SEQ ID SEQ IDSEQ ID N/A N/A NO: NO: NO: NO: 7574 7575 7576 7577 TSSNRKT RSDELRGRSDTLSA DKSTRTK SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO:NO: NO: NO: 7578 7579 7580 7581 7582 7583 DRSTRTK QSGNLHV QNAHRKTQSANRTK TSGSLSR FYMQLSR

TABLE 8 Zinc finger target site of soybean selected genomic loci LocuspDAB ID Name Number ZFP Number and Binding Sites (5′ → 3′) OGL01soy_ogl_308 124201 391 SEQ ID NO: 7363 SEQ ID NO: 7364 TACTATTCCTAAGTGGTACTAGGGGAT TTAAA AAAG OGL02 soy_ogl_307 124221 411 SEQ ID NO: 7365SEQ ID NO: 7366 GGAGGAATTTAG TACTTGCTGGTA ATAC OGL03 soy_ogl_2063 125305651 SEQ ID NO: 7367 SEQ ID NO: 7368 ATCATCTGCAAA CTTGAATTCCTATGGA OGL04soy_ogl_1906 125309 655 SEQ ID NO: 7369 SEQ ID NO: 7370 AACTTGTGAGTAATTGCATAATAA AACTGC OGL05 soy_ogl_262 124884 195 SEQ ID NO: 7371SEQ ID NO: 7372 GTTGTCTTGCTGC ACACAGGGTATCTTC TAT GAT OGL06 soy_ogl_5227124234 424 SEQ ID NO: 7373  SEQ ID NO: 7374 ATGTACTCATATT GGAGTAAGGGAAAACAT AGAT OGL07 soy_ogl_4074 124257 447 SEQ ID NO: 7375 SEQ ID NO: 7376GCTCGTCATTGAA GAAAAATAATTAATAC TTGTGTA OGL08 soy_ogl_3481 125316 662SEQ ID NO: 7377 SEQ ID NO: 7378 GGATATATAAAC ATAATGGAACCC GATGAA OGL09soy_ogl_1016 124265 455 SEQ ID NO: 7379 SEQ ID NO: 7380 GACGATCACCTCCCGGTGTCAGAGAG GAA GGCC OGL10 soy_ogl_937 124273 463 SEQ ID NO: 7381SEQ ID NO: 7382 AATGAGAGAGAG CAGATCAATCAGGG AGAAGCA TCCC OGL11soy_ogl_5109 124888 213 SEQ ID NO: 7383 SEQ ID NO: 7384 CTCTACATGGTACGAAAGGCACCTCGTA CACTCG OGL12 soy_ogl_6801 124885 215 SEQ ID NO: 7385SEQ ID NO: 7386 ATCAGCCACGAT GTCGCCCATGTCTGA CCTGCA CTCA OGL13soy_ogl_6636 124610 480 SEQ ID NO: 7387 SEQ ID NO: 7388 CTATAGTTTTAAGTATATGGTATTGGAA TGAATTA ATT OGL14 soy_ogl_4665 124614 484SEQ ID NO: 7389 SEQ ID NO: 7390 CATCGTCTCATGCTT GATCCTACAAGTGA GAGGOGL15 soy_ogl_6189 124636 506 SEQ ID NO: 7391 SEQ ID NO: 7392TTTTCTTTCTCTTTA GGAGTAGTTAGG OGL16 soy_ogl_4222 124648 518SEQ ID NO: 7393 SEQ ID NO: 7394 AACATCTTTAACT ATAGTGGTTTTGCAT CATTGTAGTG OGL17 soy_ogl_2543 121225 233 SEQ ID NO: 7395 SEQ ID NO: 7396CACGAAAAACTA AGGTATTTCTAAGAT AATTTGCT AGG OGL18 soy_ogl_310 121227 235SEQ ID NO: 7397 SEQ ID NO: 7398 TTTGCTGAGTGAAGG CAAATGTGATAACTG ATGACOGL19 soy_ogl_2353 121233 241 SEQ ID NO: 7399 SEQ ID NO: 7400AAGATGAAGCGAGAT ATCGTTCAAGAAGTT GAA OGL20 soy_ogl_1894 121235 243SEQ ID NO: 7401 SEQ ID NO: 7402 CAGGCTGGCAAA GGGTGGTAAGTACTT ATGGAA GAAOGL22 soy_ogl_3218 121238 250 SEQ ID NO: 7403 SEQ ID NO: 7404AATGCGTGGCCA AACTAGCGTAGAGT CGAT AGAT OGL24 soy_ogl_3333 121246 259SEQ ID NO: 7405 SEQ ID NO: 7406 GAGAAAGCCATGGTC TGTGTGGAAGAC OGL25soy_ogl_2546 121249 262 SEQ ID NO: 7407 SEQ ID NO: 7408 TGGATGTCAAGTTAGGGGAGAATACAG ATTCAAG OGL28 soy_ogl_5957 125324 670 SEQ ID NO: 7409SEQ ID NO: 7410 GGGAGGGAGACCCAA GGGAGAAACAAAAAG OGL30 soy_ogl_3818121265 282 SEQ ID NO: 7411 SEQ ID NO: 7412 GTTTGGTTAGGCG GGAGAAACAACTGCAGATC OGL31 soy_ogl_5551 121271 288 SEQ ID NO: 7413 SEQ ID NO: 7414AAAGTGTCATGCC GCAATTGCGGTTGCA OGL33 optimal_loci_ 124666 538SEQ ID NO: 7415 SEQ ID NO: 7416 1098 GGTATCGTATTGC CGCACGTAATAA ATTAGOGL34 optimal_loci_ 124814 598 SEQ ID NO: 7417 SEQ ID NO: 7418 97772GAAGAAATTATTGCA AATCAGAGGGAAGT GAGA OGL35 optimal_loci_ 124690 560SEQ ID NO: 7419 SEQ ID NO: 7420 236662 AACTAACTTGTA TTGGCGGGAATTAGTACAACTG AGA OGL36 optimal_loci_ 124815 599  SEQ ID NO: 7421SEQ ID NO: 7422 139485 GATCTTGCGGGG GACCTGGTGGTCATG TAGCAG OGL37optimal_loci_ 125338 627 SEQ ID NO:7584 OGL37 301175 ATACGTCAGGGTtantgGTTGTTTAAT GAAAAGCC OGL38 optimal_loci_ 124816 600 SEQ ID NO: 7423SEQ ID NO: 7424 152337 TCTATGTCGGACTTT GATCATTTAAGGATAA OGL39optimal_loci_ 124842 631 SEQ ID NO: 7425 SEQ ID NO: 7426 202616ATGAATTCCCTTT TTTGCTGCTTTATAG TCTTA

The soybean representative genomic loci zinc finger designs wereincorporated into zinc finger expression vectors encoding a proteinhaving at least one finger with a CCHC structure. See, U.S. PatentPublication No. 2008/0182332. In particular, the last finger in eachprotein had a CCHC backbone for the recognition helix. The non-canonicalzinc finger-encoding sequences were fused to the nuclease domain of thetype 11S restriction enzyme FokI (amino acids 384-579 of the sequence ofWah et al., (1998) Proc. Natl. Acad. Sci. USA 95:10564-10569) via a fouramino acid ZC linker and an opaque-2 nuclear localization signaloptimized for soybean to form zinc-finger nucleases (ZFNs). See, U.S.Pat. No. 7,888,121. Zinc fingers for the various functional domains wereselected for in vivo use. Of the numerous ZFNs that were designed,produced and tested to bind to the putative genomic target site, theZFNs described in Table 8 above were identified as having in vivoactivity and were characterized as being capable of efficiently bindingand cleaving the unique soybean genomic polynucleotide target sites inplanta.

ZFN Construct Assembly

Plasmid vectors containing ZFN gene expression constructs were designedand completed using skills and techniques commonly known in the art(see, for example, Ausubel or Maniatis). Each ZFN-encoding sequence wasfused to a sequence encoding an opaque-2 nuclear localization signal(Maddaloni et al., (1989) Nuc. Acids Res. 17:7532), that was positionedupstream of the zinc finger nuclease. The non-canonical zincfinger-encoding sequences were fused to the nuclease domain of the type11S restriction enzyme FokI (amino acids 384-579 of the sequence of Wahet al. (1998) Proc. Natl. Acad. Sci. USA 95:10564-10569). Expression ofthe fusion proteins was driven by a strong constitutive promoter fromthe Cassava vein mosaic virus. The expression cassette also includes a3′ UTR from the Agrobacterium tumefaciens ORF23. The self-hydrolyzing 2Aencoding the nucleotide sequence from Thosea asigna virus (Szymczak etal., (2004) Nat. Biotechnol. 22:760-760) was added between the two ZincFinger Nuclease fusion proteins that were cloned into the construct.

The plasmid vectors were assembled using the IN-FUSION™ AdvantageTechnology (Clontech, Mountain View, Calif.). Restriction endonucleaseswere obtained from New England BioLabs (Ipswich, Mass.) and T4 DNALigase (Invitrogen, Carlsbad, Calif.) was used for DNA ligation. Plasmidpreparations were performed using NUCLEOSPIN® Plasmid Kit(Macherey-Nagel Inc., Bethlehem, Pa.) or the Plasmid Midi Kit (Qiagen)following the instructions of the suppliers. DNA fragments were isolatedusing QIAQUICK GEL EXTRACTION KIT™ (Qiagen) after agarose tris-acetategel electrophoresis. Colonies of all ligation reactions were initiallyscreened by restriction digestion of miniprep DNA. Plasmid DNA ofselected clones was sequenced by a commercial sequencing vendor(Eurofins MWG Operon, Huntsville, Ala.). Sequence data were assembledand analyzed using the SEQUENCHER™ software (Gene Codes Corp., AnnArbor, Mich.). Plasmids were constructed and confirmed via restrictionenzyme digestion and via DNA sequencing.

Zinc Finger Cloning Via Automated Workflow

A subset of Zinc Finger Nuclease vectors were cloned via an automatedDNA construction pipeline. Overall, the automted pipeline resulted invector constructions with identical ZFN architecture as describedpreviously. Each Zinc Finger monomer, which confers the DNA bindingspecificity of the ZFN, were divided into 2-3 unique sequences at a KPFamino acid motif. Both the 5′ and 3′ ends of the ZFN fragments weremodified with inclusion of a BsaI recognition site (GGTCTCN) and derivedoverhangs. Overhangs were distributed such that a 6-8 part assemblywould only result in the desired full length expression clone. ModifiedDNA fragments were synthesized de novo (Synthetic Genomics Incorporated,La Jolla, Calif.). A single dicot backbone, pDAB118796 was used in allof the soybean ZFN builds. It contained the Cassava Mosaic Viruspromoter and the Opaque2 NLS as well as the FokI domain and the Orf233′UTR from Agrobacterium tumefaciens. Cloned in between the Opaque 2 NLSand the FokI domain was a BsaI flanked SacB gene from Bacillus subtilis.When putative ligation events were plated on Sucrose containing media,the SacB cassette acts as a negative selection agent reducing oreliminating vector backbone contamination. A second part repeatedlyutilized in all builds was pDAB117443. This vector contains the firstmonomer FokI domain, the T2A stutter sequence, and the 2^(nd) monomerOpaque2 NLS all flanked by BsaI sites.

Using these materials as the ZFN DNA parts library, a Freedom Evo 150®(TECAN, Mannedorf, Switzerland) manipulated the addition of 75-100 ng ofeach DNA plasmid or synthesized fragment from 2D bar coded tubes into aPCR plate (ThermoFisher, Waltham, Mass.). BsaI (NEB, Ipswich, Mass.) andT4 DNA ligase (NEB, Ipswich, Mass.) supplemented with Bovine SerumAlbumin protein (NEB, Ipswich, Mass.) and T4 DNA Ligase Buffer (NEB,Ipswich, Mass.) were added to the reaction. Reactions were cylcled (25×)with incubations for 3 minutes at 37° C. and 4 minutes at 16° C. C1000Touch Thermo Cycler® (BioRad, Hercules Calif.). Ligated material wastransformed and screened in Top10 Cells® (Life Technologies Carlsbad,Calif.) by hand or using a Qpix460 colony picker and LabChip GX® (PerkinElmer, Waltham, Mass.). Correctly digesting colonies were sequenceconfirmed provided to plant transformation.

Universal Donor Construct Assembly

To support rapid testing of a large number of target loci, a novel,flexible universal donor system sequence was designed and constructed.The universal donor polynucleotide sequence was compatible with highthroughput vector construction methodologies and analysis. The universaldonor system was composed of at least three modular domains: a variableZFN binding domain, a non-variable analytical and user defined featuresdomain, and a simple plasmid backbone for vector scale up. Thenon-variable universal donor polynucleotide sequence was common to alldonors and permits design of a finite set of assays that can be usedacross all of the soybean target sites thus providing uniformity intargeting assessment and reducing analytical cycle times. The modularnature of these domains allowed for high throughput donor assembly.Additionally, the universal donor polynucleotide sequence has otherunique features aimed at simplifying downstream analysis and enhancingthe interpretation of results. It contained an asymmetric restrictionsite sequence that allows the digestion of PCR products intodiagnostically predicted sizes. Sequences comprising secondarystructures that were expected to be problematic in PCR amplificationwere removed. The universal donor polynucleotide sequence was small insize (less than 3.0 Kb). Finally, the universal donor polynucleotidesequence was built upon the high copy pUC19 backbone that allows a largeamount of test DNA to be bulked in a timely fashion.

As an embodiment, an example plasmid comprising a universal donorpolynucleotide sequence is provided as pDAB124280 (SEQ ID NO:7561 andFIG. 7). In an additional embodiment, a universal donor polynucleotidesequence is provided as: pDAB124281, SEQ ID NO:7562, FIG. 8; pDAB121278,SEQ ID NO:7563, FIG. 9; pDAB123812, SEQ ID NO:7564 FIG. 10; pDAB121937,SEQ ID NO:7565, FIG. 11; pDAB123811, SEQ ID NO:7566, FIG. 12; and,pDAB124864 SEQ ID NO:7567, FIG. 13. In another embodiment, additionalsequences comprising the universal donor polynucleotide sequence withfunctionally expressing coding sequence or nonfunctional (promoterless)expressing coding sequences can be constructed (Table 11).

TABLE 11 The various universal domain sequences that were transformedinto the plant cell protoplasts for donor mediated integration withinthe genome of soybean are provided. The various elements of theuniversal domain plasmid system are described and identified by basepair position in the accompanying SEQ ID NO:. “N/A” meas not applicable.SEQ ZFN binding Analytical Plasmid ID Vector Name domain domain backboneNO: pDAB124280 1955-2312 bp 2313-2422 bp 2423-1954 bp 7561 pDAB1242811955-2256 bp 2257-2366 bp 2367-1954 bp 7562 pDAB121278 1509-1724 bp1725-1834 bp 1835-1508 bp 7563 pDAB123812 1955-2177 bp 2178-2287 bp2288-1954 bp 7564 pDAB121937 1955-2127 bp 2128-2237 bp 2238-1954 bp 7565pDAB123811 1955-2187 bp 2288-2297 bp 2298-1954 bp 7566 pDAB1248641952-2185 N/A 2186-1951 bp 7567

The universal donor polynucleotide sequence was a small 2-3 Kb modulardonor system delivered as a plasmid. This was a minimal donor,comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or more ZFN binding sites, a short100-150 bp template region referred to as “DNA X” or “UZI Sequence” (SEQID NO:7568) that carries restriction sites and DNA sequences for primerdesign or coding sequences, and a simple plasmid backbone (FIG. 4). Theentire plasmid was inserted through NHEJ following DNA double strandbreaks at the appropriate ZFN binding site; the ZFN binding sites can beincorporated tandemly. This embodiment of a universal donorpolynucleotide sequence was most suitable for rapid screening of targetsites and ZFNs, and sequences that were difficult to amplify wereminimized in the donor. Universal donors without the “UZI” sequence butcarrying one or more ZFN sites have also been generated

In a further embodiment the universal donor polynucleotide sequence wasmade up of at least 4 modules and carries ZFN binding sites, homologyarms, DNA X with either just the approximately 100 bp analytical pieceor coding sequences. This embodiment of the universal donorpolynucleotide sequence was suitable for interrogating HDR mediated geneinsertion at a variety of target sites, with several ZFNs (FIG. 5).

The universal donor polynucleotide sequence can be used with alltargeting molecules with defined DNA binding domains, with two modes oftargeted donor insertion (NHEJ/HDR). As such, when the universal donorpolynucleotide sequence was co-delivered with the appropriate ZFNexpression construct, the donor vector and the soybean genome was cut inone specific location dictated by the binding of the particular ZFN.Once linearized, the donor can be incorporated into the genome by NHEJor HDR. The different analytical considerations in the vector design canthen be exploited to determine the Zinc Finger which maximizes theefficient delivery of targeted integration.

Example 4: Soybean Transformation Procedures

Before delivery to Glycine max c.v. Maverick protoplasts, plasmid DNAfor each ZFN construct was prepared from cultures of E. coli using thePURE YIELD PLASMID MAXIPREP SYSTEM® (Promega Corporation, Madison, Wis.)or PLASMID MAXI KIT® (Qiagen, Valencia, Calif.) following theinstructions of the suppliers.

Protoplast Isolation

Protoplasts were isolated from a Maverick suspension culture derivedfrom callus produced from leaf explants. Suspensions were subculturedevery 7 days in fresh LS medium (Linsmaier and Skoog 1965) containing 3%(w/v) sucrose, 0.5 mg/L 2,4-D, and 7 g of bactoagar, pH 5.7 Forisolation, thirty milliliters of a Maverick suspension culture 7 dayspost subculturing was transferred to a 50 ml conical tube andcentrifuged at 200 g for 3 minutes, yielding about 10 ml of settled cellvolume (SCV) per tube. The supernatant was removed and twentymilliliters of the enzyme solution (0.3% pectolyase (320952; MPBiomedicals), 3% cellulase (“Onozuka” R10™; Yakult Pharmaceuticals,Japan) in MMG solution (4 mM MES, 0.6 M mannitol, 15 mM MgCl₂, pH 6.0)was added for every 4 SCV of suspension cells and the tubes were wrappedwith Parafilm™. The tubes were placed on a platform rocker overnight(about 16-18 hr) and an aliquot of the digested cells was viewedmicroscopically to ensure the digestion of the cell wall was sufficient.

Protoplast Purification

A soybean (Glycine max c.v. Maverick) protoplast-based transformationmethod was developed and used for soybean protoplast transformation.Protoplasts were isolated from a Maverick suspension culture derivedfrom callus produced from leaf explants. The techniques provided belowdescribe the method. Soybean cell suspensions were subcultured every 7days by a 1:5 dilution in fresh LS medium (Linsmaier and Skoog 1965)containing 3% (w/v) sucrose, 0.5 mg/L 2,4-D, and 7 g of bactoagar, pH5.7 All experiments were performed starting 7 days post subculture basedon the protocol described below.

Protoplast Isolation

Thirty milliliters of a soybean c.v. Maverick suspension culture 7 dayspost subculturing was transferred to a 50 ml conical centrifuge tube andcentrifuged at 200 g for 3 minutes, yielding about 10 ml of settled cellvolume (SCV) per tube. The supernatant was removed without disturbingthe cell pellet. Twenty milliliters of the enzyme solution (0.3%pectolyase (320952; MP Biomedicals), 3% cellulase (“Onozuka” R10™;Yakult Pharmaceuticals, Japan) in MMG solution (4 mM MES, 0.6 Mmannitol, 15 mM MgCl₂, pH 6.0) was added for every 4 SCV of suspensioncells and the tubes were wrapped with Parafilm™. The tubes were placedon a platform rocker overnight (about 16-18 hr). The next morning, analiquot of the digested cells was viewed microscopically to ensure thedigestion of the cell walls was sufficient.

Protoplast Purification

The cells/enzyme solutions were filtered slowly through a 100 μM cellstrainer. The cell strainer was rinsed with 10 ml of W5+ media (1.82 mMMES, 192 mM NaCl, 154 mM CaCl₂, 4.7 mM KCl, pH 6.0). The filtering stepwas repeated using a 70 μM screen. The final volume was brought to 40 mlby adding 10 ml of W5+ media. The cells were mixed by inverting thetube. The protoplasts were slowly layered onto 8 ml of a sucrose cushionsolution (500 mM sucrose, 1 mM CaCl₂, 5 mM MES-KOH, pH 6.0) by addingthe cushion solution to the bottom of a 50 ml conical centrifuge tubecontaining the cells. The tubes were centrifuged at 350×g for 15 minutesin a swinging bucket rotor. A 5 ml pipette tip was used to slowly removethe protoplast band (about 7-8 ml). The protoplasts were thentransferred to a 50 ml conical tube and 25 ml of W5+ wash was added. Thetubes were inverted slowly and the centrifuged for 10 minutes at 200 g.The supernatant was removed, 10 ml of MMG solution was added and thetube was inverted slowly to resuspend the protoplasts. The protoplastdensity was determined using a haemocytometer or a flow cytometer.Typically, 4 PCV of cells suspension yields about 2 million protoplasts.

Transformation of Protoplasts using PEG

The protoplast concentration was adjusted to 1.6 million/ml with MMG.Protoplast aliquots of 300 μl (about 500,000 protoplasts) weretransferred into 2 ml sterile tubes. The protoplast suspension was mixedregularly during the transfer of protoplasts into the tubes. Plasmid DNAwas added to the protoplast aliquots according to the experimentaldesign. The rack containing the tubes of protoplasts was slowly inverted3 times for 1 minute each to mix the DNA and protoplasts. Theprotoplasts were incubated for 5 minutes at room temperature. Threehundred microliters of a polyethlene glycol (PEG 4000) solution (40%ethylene glycol (81240-Sigma Aldrich), 0.3 M mannitol, 0.4 M CaCl₂) wasadded to the protoplasts and the rack of tubes was mixed for 1 min andincubated for 5 min, with gentle inversion twice during the incubation.One milliliter of W5+ was slowly added to the tubes and the rack oftubes inverted 15-20 times. The tubes were then centrifuged at 350 g for5 min and the supernatant removed without disturbing the pellet. Onemilliliter of WI media (4 mM MES 0.6 M mannitol, 20 mM KCl, pH 6.0) wasadded to each tube and the rack was gently inverted to resuspend thepellets. The rack was covered with aluminum foil and laid on its side toincubate overnight at 23° C.

Measuring Transformation Frequency and Harvesting the Protoplasts

Quantification of protoplasts and transformation efficiencies weremeasured using a Quanta Flow Cytometer™ (Beckman-Coulter Inc).Approximately 16-18 hours post transformation, 100 μl from eachreplicate was sampled, placed in a 96 well plate and diluted 1:1 with WIsolution. The replicates were resuspended 3 times and 100 μl wasquantified using flow cytometry. Prior to submitting the samples foranalysis, the samples were centrifuged at 200 g for 5 min, supernatantswere removed and the samples were flash frozen in liquid nitrogen. Thesamples were then placed in a −80° C. freezer until processing formolecular analysis.

Transformation of ZFN and Donor

For each of the selected genomic loci of Table 5, the soybeanprotoplasts were transfected with constructs comprising a greenfluorescent protein (gfp) gene expressing control, ZFN alone, donoralone and a mixture of ZFN and donor DNA at a ratio of 1:10 (by weight).The total amount of DNA for transfection of 0.5 million protoplasts was80 μg. All treatments were conducted in replicates of three. The gfpgene expressing control used was pDAB7221 (FIG. 14, SEQ ID NO:7569)containing the Cassava Vein Mosaic Virs promoter—green fluorescentprotein coding sequence—Agrobacterium tumefaciens ORF24 3′UTR geneexpression cassettes. To provide a consistent amount of total DNA pertransfection, either salmon sperm or a plasmid containing a gfp gene wasused as filler where necessary. In a typical targeting experiment, 4 μgof ZFN alone or with 36 μg of donor plasmids were transfected and anappropriate amount of salmon sperm or pUC19 plasmid DNA was added tobring the overall amount of DNA to the final amount of 80 μg. Inclusionof gfp gene expressing plasmid as filler allows assessment oftransfection quality across multiple loci and replicate treatments.

Example 5: Cleavage of Genomic Loci in Soybean Via Zinc Finger Nuclease

Targeting at select genomic loci was demonstrated by ZFN induced DNAcleavage and donor insertion using the protoplast based Rapid TargetingSystem (RTA). For each soybean select locus, up to six ZFN designs weregenerated and transformed into protoplasts either alone or with auniversal donor polynucleotide and ZFN mediated clevage and insertionwas measured using Next Generation Sequencing (NGS) or junctional(in-out) PCR respectively.

ZFN transfected soybean protoplasts were harvested 24 hourspost-transfection, by centrifugation at 1600 rpm in 2 ml EPPENDORF™tubes and the supernatant was completely removed. Genomic DNA wasextracted from protoplast pellets using the QIAGEN PLANT DNA EXTRACTIONKIT™ (Qiagen, Valencia, Calif.). The isolated DNA was resuspended in 50μL of water and concentration was determined by NANODROP® (Invitrogen,Grand Island, N.Y.). The integrity of the DNA was estimated by runningsamples on 0.8% agarose gel electrophoresis. All samples were normalized(20-25 ng/μL) for PCR amplification to generate amplicons for sequencing(Illumina, Inc., SanDiego, Calif.). Bar-coded PCR primers for amplifyingregions encompassing each test ZFN recognition sequence from treated andcontrol samples were designed and purchased from IDT (Coralville, Iowa,HPLC purified). Optimum amplification conditions were identified bygradient PCR using 0.2 μMappropriate bar-coded primers, ACCUPRIME PFXSUPERMIX™ (Invitrogen, Carlsbad, Calif.) and 100 ng of template genomicDNA in a 23.5 μL reaction. Cycling parameters were initial denaturationat 95° C. (5 min) followed by 35 cycles of denaturation (95° C., 15sec), annealing (55-72° C., 30 sec), extension (68° C., 1 min) and afinal extension (68° C., 7 min). Amplification products were analyzed on3.5% TAE agarose gels and appropriate annealing temperature for eachprimer combination was determined and used to amplify amplicons fromcontrol and ZFN treated samples as described above. All amplicons werepurified on 3.5% agarose gels, eluted in water and concentrations weredetermined by NANODROP™. For Next Generation Sequencing 100 ng of PCRamplicon from the ZFN treated and corresponding untreated controls werepooled together and sequenced using llumina Next Generation Sequencing(NGS).

The cleavage activity of appropriate ZFNs at each soybean optimalgenomic loci were assayed. Short amplicons encompassing the ZFN cleavagesites were amplified from the genomic DNA and subjected to IIlumina NGSfrom ZFN treated and control protoplasts. The ZFN induced cleavage orDNA double strand break was resolved by the cellular NHEJ repair pathwayby insertion or deletion of nucleotides (indels) at the cleavage siteand presence of indels at the cleavage site was thus a measure of ZFNactivity and was determined by NGS. Cleavage activity of the targetspecific ZFNs was estimated as the number of sequences with indels per 1million high quality sequences using NGS analysis software (Patentpublication 2012-0173,153, data Analysis of DNA sequences). Activitieswere observed for sobyean selected genomic loci targets and were furtherconfirmed by sequence alignments that show a diverse footprint of indelsat each ZFN cleavage site. This data suggests that the soybean selectedgenomic loci were amenable to cleavage by ZFNs. Differential activity ateach target was reflective of its chromatin state and amenability tocleavage as well as the efficiency of expression of each ZFN.

Example 6: Rapid Targeting Analysis of the Integration of aPolynucleotide Donor

Validation of the targeting of the universal donor polynucleotidesequence within the soybean selected genomic loci targets vianon-homologous end joining (NHEJ) meditated donor insertion, wasperformed using a semi-throughput protoplast based Rapid TargetingAnalysis method. For each soybean selected genomic loci target, around3-6 ZFN designs were tested and targeting was assessed by measuring ZFNmediated cleavage by Next Generation Sequencing methods and donorinsertion by junctional in-out PCR (FIG. 6). Soybean selected genomicloci that were positive in both assays were identified as a targetablelocus.

ZFN Donor Insertion Rapid Targeting Analysis

To determine if a soybean selected genomic loci target can be targetedfor donor insertion, a ZFN construct and universal donor polynucleotideconstruct were co-delivered to soybean protoplasts which were incubatedfor 24 hours before the genomic DNA was extracted for analysis. If theexpressed ZFN was able to cut the target binding site both at thesoybean selected genomic loci target and in the donor, the linearizeddonor would then be inserted into the cleaved target site in the soybeangenome via the non-homologous end joining (NHEJ) pathway. Confirmationof targeted integration at the soybean selected genomic loci target wascompleted based on an “In-Out” PCR strategy, where an “In” primerrecognizes sequence at the native optimal genomic loci and an “Out”primer binds to sequence within the donor DNA. The primers were designedin a way that only when the donor DNA was inserted at the soybeanselected genomic loci target, would the PCR assay produce anamplification product with the expected size. The In-Out PCR assay wasperformed at both the 5′- and 3′-ends of the insertion junction. Theprimers used for the analysis of integrated polynucleotide donorsequences are provided in Table 9.

ZFN Donor insertion at Target Loci using nested “In-Out” PCR

All PCR amplifications were conducted using a TAKARA EX TAQ HS™ kit(Clonetech, Mountain View, Calif.). The first In-Out PCR was carried outin 25 μL final reaction volume that contains 1× TAKARA EX TAQ HS™buffer, 0.2 mM dNTPs, 0.2 μM “Out” primer, 0.05 μM “In” primer (designedfrom the universal donor cassette described above), 0.75 unit of TAKARAEX TAQ HS™ polymerase, and 6 ng extracted soybean protoplast DNA. Thereaction was then completed using a PCR program that consists of 94° C.for 3 min, 14 cycles of 98° C. for 12 sec, and 60° C. for 30 sec, and72° C. for 1 min, followed by 72° C. for 10 min and held at 4° C. FinalPCR products were run on an agarose gel along with 1 KB PLUS DNA LADDER™(Life Technologies, Grand Island, N.Y.) for visualization.

The nested In-Out PCR was conducted in 25 μL final reaction volume thatcontained 1× TAKARA EX TAQ HS™ buffer, 0.2 mM dNTPs, 0.2 μM “Out” primer(Table 9), 0.1 μM “In” primer (designed from the universal donorcassette described above, Table 10), 0.75 units of TAKARA EX TAQ HS™polymerase, and 1 μL of the first PCR product. The reaction was thencompleted using a PCR program that consisted of 94° C. for 3 min, 30cycles of 98° C. for 12 sec, 60° C. for 30 sec and 72° C. for 45 sec,followed by 72° C. for 10 min and held at 4° C. Final PCR products wererun on an agarose gel along with 1 KB PLUS DNA LADDER™ (LifeTechnologies, Grand Island, N.Y.) for visualization.

TABLE 9 List of all ″Out″primers for nested In-Out PCR analysis of optimal genomic loci. OGL01First PCR 5′-end MAS1057 SEQ ID NO: 7427 CAAACAAGGAGAGAGCGAG GM SpecSEQ ID NO: 7428 GATCGACATTGATCTGGCTA 3′-end MAS1059 SEQ ID NO: 7429GGCAAGGACACAAACGG GM Uzi SEQ ID NO: 7430 ATATGTGTCCTACCGTATCAGG Nest PCR5′-end MAS1058 SEQ ID NO: 7431 TACCCAAGAAGAAACATTAGACC GM SpecSEQ ID NO: 7432 Nst GTTGCCTTGGTAGGTCC 3′-end MAS1060 SEQ ID NO: 7433ATGTAGTTGTTTCTCTGCTGTG GM Uzi Nst SEQ ID NO: 7434 GAGCCATCAGTCCAACACOGL02 First PCR 5′-end MAS1061 SEQ ID NO: 7435 CACGAGGTTTACGCCAT 3′-endMAS1063 SEQ ID NO: 7436 TCTGATAACTTGCTAGTGTGTG Nest PCR 5′-end MAS1062SEQ ID NO: 7437 GCTGCTCAGTGGATGTC 3′-end MAS1064 SEQ ID NO: 7438TCGTTTATCGGGATTGTCTC OGL03 First PCR 5′-end MAS1133 SEQ ID NO: 7439TTGTTGCTTCTATGCTCCTC 3′-end MAS1135 SEQ ID NO: 7440 CGTCGTTGTGGATGAGGNest PCR 5′-end MAS1134 SEQ ID NO: 7441 CCATTGCTGTTCTGCTTG 3′-endMAS1136 SEQ ID NO: 7442 TGTAGGTGACGGGTGTG OGL04 First PCR 5′-end MAS1155SEQ ID NO: 7443 GTGTGTTATTGTCTGTGTTCTC 3′-end MAS1139 SEQ ID NO: 7444GACTCCTATGTGCCTGATTC Nest PCR 5′-end MAS1156 SEQ ID NO: 7445GAGAACGATGGATAGAAAAGCA 3′-end MAS1140 SEQ ID NO: 7446TTTGTTTCAGTCTTGCTCCT OGL05 First PCR 5′-end MAS1121 SEQ ID NO: 7447CTACCTATAAACTGGACGGAC 3′-end MAS1123 SEQ ID NO: 7448CGTCAAATGCCCATTATTCAT Nest PCR 5′-end MAS1122 SEQ ID NO: 7449GATTTGGGCTTGGGCATA 3′-end MAS1124 SEQ ID NO: 7450 TGAATCCCACTAGCACCATOGL06 First PCR 5′-end MAS1065 SEQ ID NO: 7451 GGAGATAGAGTTAGAAGGTTTTGA3′-end MAS1067 SEQ ID NO: 7452 GAGGTTGTTTTGACGCCA Nest PCR 5′-endMAS1066 SEQ ID NO: 7453 AAGGAAGAAATGTGAAAAAGAAGAC 3′-end MAS1068SEQ ID NO: 7454 AGAGAAGCGAAACCCAAAG OGL07 First PCR 5′-end MAS1069SEQ ID NO: 7455 GACCCATTTATCTATCCCGTAT 3′-end MAS1071 SEQ ID NO: 7456GGCTCGTATCAGTTCCATTTAG Nest PCR 5′-end MAS1070 SEQ ID NO: 7457AAGTACGAACAAGATTGGTGAG 3′-end MAS1072 SEQ ID NO: 7458TCTATTACATTCCATCCAAAGGC OGL08 First PCR 5′-end MAS1141 SEQ ID NO: 7459GAAACGAGAGAGATGACCAATA 3′-end MAS1143 SEQ ID NO: 7460 GGTTCACGGGTTCAGCNest PCR 5′-end MAS1142 SEQ ID NO: 7461 CCTGACGCAAAAGAAGAAATG 3′-endMAS1144 SEQ ID NO: 7462 GTTATACTTACTGTCACCACGAG OGL09 First PCR 5′-endMAS1073 SEQ ID NO: 7463 TTATTCCTGCGTCTCTCAC 3′-end MAS1075SEQ ID NO: 7464 TTGTGCGTGATAAATAGGGC Nest PCR 5′-end MAS1074SEQ ID NO: 7465 GATAGTTGATTGTGTTGTTAGCATA 3′-end MAS1076 SEQ ID NO: 7466CTCACCTGTTGCCCGTA OGL10 First PCR 5′-end MAS1077 SEQ ID NO: 7467GTTTGAGTTGGCAGGTGT 3′-end MAS1079 SEQ ID NO: 7468 CCGTGACTTGTGCTAGAGNest PCR 5′-end MAS1078 SEQ ID NO: 7469 CTGAAGTTGACGCCGC 3′-end MAS1080SEQ ID NO: 7470 AAGCACAGGACGGTTAGA OGL11 First PCR 5′-end MAS1125SEQ ID NO: 7471 CACGGGTCACAAATCTAGTT 3′-end MAS1127 SEQ ID NO: 7472CCATTAAGTCTGTCTCAACTTTC Nest PCR 5′-end MAS1126 SEQ ID NO: 7473CTGCTTGAGTAGGAAGAAGTG 3′-end MAS1128 SEQ ID NO: 7474 ATCACCAAAGCCGAGAACOGL12 First PCR 5′-end MAS1129 SEQ ID NO: 7475 GTAGGCGTGAAGGCTG 3′-endMAS1131 SEQ ID NO: 7476 TGAAACCGCACAATCTCG Nest PCR 5′-end MAS1130SEQ ID NO: 7477 CCCTCCGAAACAATCCG 3′-end MAS1132 SEQ ID NO: 7478ACCCGTTGAATGCGAG OGL13 First PCR 5′-end MAS1081 SEQ ID NO: 7479AAGGTGGATGGGAGGAA 3′-end MAS1083 SEQ ID NO: 7480TGGCACTAATACATTACATAAGACT Nest PCR 5′-end MAS1082 SEQ ID NO: 7481ATGTTACTTCAATCCCTCGTC 3′-end MAS1084 SEQ ID NO: 7482TGAATAGGGCAAAAACACAC OGL14 First PCR 5′-end MAS1085 SEQ ID NO: 7483CAAGTGAGCAGGGCG 3′-end MAS1087 SEQ ID NO: 7484 CTATCATTCGTAAAGTTTGAGGACNest PCR 5′-end MAS1086 SEQ ID NO: 7485 AGCCTCACTCACAACAAAG 3′-endMAS1088 SEQ ID NO: 7486 TGAAACTGTCTTGTGACTTACC OGL15 First PCR 5′-endMAS1089 SEQ ID NO: 7487 GCACTGACATACCAACAATC 3′-end MAS1091SEQ ID NO: 7488 GTTGTCGGGATTTCACTTCAT Nest PCR 5′-end MAS1090SEQ ID NO: 7489 GATAGGAGAAAGAGCAAGGAC 3′-end  MAS1092 SEQ ID NO: 7490TTCTCAACATCAACTCATACACTC OGL16 First PCR 5′-end MAS1093 SEQ ID NO: 7491CTCAAAGCAACATCAACCAT 3′-end MAS1095 SEQ ID NO: 7492 AATCCCAAAGCAGCCAACNest PCR 5′-end MAS1094 SEQ ID NO: 7493 AAACACAAATCACATCATAGTAAAC 3′-endMAS1096 SEQ ID NO: 7494 GCTAGTATGCTTCTGTCAGTTTA OGL17 First PCR 5′-endMAS916 SEQ ID NO: 7495 ACTAGTTCTTTCCCGAACATT 3′-end MAS918SEQ ID NO: 7496 CATTTGGTGATTTAACTCATCAGC Nest PCR 5′-end MAS917SEQ ID NO: 7497 AAATTTACCACGGTTGGTCC 3′-end MAS919 SEQ ID NO: 7498TCTGCATTAACTATATCAGGAGG OGL18 First PCR 5′-end MAS920 SEQ ID NO: 7499ATTCAACATTTACCCTTCACAA 3′-end MAS922 SEQ ID NO: 7500AATTCTTTCTCATACTTGGTTGT Nest PCR 5′-end MAS921 SEQ ID NO: 7501CCTTGTTTTCCGTACTATCAATT 3′-end MAS923 SEQ ID NO: 7502TATTGGAGTAATGTGGACAAGC OGL19 First PCR 5′-end MAS924 SEQ ID NO: 7503AACAACTTTCCAACCCACAA 3′-end  MAS1009 SEQ ID NO: 7504CGTTTTACCTTGACTTGACCT Nest PCR 5′-end MAS925 SEQ ID NO: 7505CCAGAGAGGAACCAGAAGT 3′-end MAS1010 SEQ ID NO: 7506 CCTTAGACAAAACTCGCACTTOGL20 First PCR 5′-end MAS1011 SEQ ID NO: 7507 GAAAGAGAAGACGCCACC 3′-endMAS930 SEQ ID NO: 7508 TCATTAGAGGGTCAAAAGTGC Nest PCR 5′-end MAS1012SEQ ID NO: 7509 CCTGAAGAAAAGTGGGAGAA 3′-end MAS931 SEQ ID NO: 7510TTCAATCATAATTAAACTAATAA GACTGT OGL22 First PCR 5′-end MAS960SEQ ID NO: 7511 ACTGAATGTATTGTCCGACG 3′-end MAS962 SEQ ID NO: 7512GCCCTACATTTTCATTTCATTGG Nest PCR 5′-end MAS961 SEQ ID NO: 7513GTGAGACCGCCCCTT 3′-end MAS963 SEQ ID NO: 7514 CCACTACTTTTTACTCACAGAAGAOGL24 First PCR 5′-end MAS968 SEQ ID NO: 7515 GTCAATTCTCATCAGTTCCATCT3′-end MAS970 SEQ ID NO: 7516 CGATGAATAGTATGAGTGCGTAG Nest PCR 5′-endMAS969 SEQ ID NO: 7517 TGCGTCTCTTGCTTCCTA 3′-end MAS971 SEQ ID NO: 7518GCCACGAGAGGATAGAATAAT OGL25 First PCR 5′-end MAS972 SEQ ID NO: 7519TAGTGTACCCTCCTCATCATA 3′-end MAS974 SEQ ID NO: 7520GATAATCAAATGAGTGGACGAATA Nest PCR 5′-end MAS973 SEQ ID NO: 7521TGTATTTGGATAAGTGTGGGAC 3′-end MAS975 SEQ ID NO: 7522GATTTTAGCGTGATTGATGGAAG OGL28 First PCR 5′-end MAS1149 SEQ ID NO: 7523CTGAAGCAAGTGGTGATGTT 3′-end MAS1151 SEQ ID NO: 7524 CTTACCACCACCTGCGNest PCR 5′-end MAS1150 SEQ ID NO: 7525 GCATAAAGGTCAGCAGAGG 3′-endMAS1152 SEQ ID NO: 7526 TACTCTTTAGCCATAGCCAAT OGL30 First PCR 5′-endMAS988 SEQ ID NO: 7527 GTTTATTGCCAGAGACGGT 3′-end MAS990 SEQ ID NO: 7528CGTCGTTGCTTGCTTGT Nest PCR 5′-end MAS989 SEQ ID NO: 7529GGAAAGACATAAAAGTAAATGGAAG 3′-end MAS991 SEQ ID NO: 7530TAACTACCTGATAACCTCCTTTT OGL31 First PCR 5′-end MAS992 SEQ ID NO: 7531GCAAACTTTAAGTAAACTAGAGGC 3′-end MAS994 SEQ ID NO: 7532AGTGTACTCTAGTCAGATTTTGC Nest PCR 5′-end MAS993 SEQ ID NO: 7533CAACCCAACAAGCAAACAC 3′-end MAS995 SEQ ID NO: 7534CTCGGTTTTGTAGTCATCTATGTA OGL33 First PCR 5′-end MAS1101 SEQ ID NO: 7535GATGAATAACAGTGCGAGGA 3′-end MAS942 SEQ ID NO: 7536 CTGTAATCCTCATTTTGCACGNest PCR 5′-end MAS941 SEQ ID NO: 7537 GGGGTAGTTACACTTCTGC 3′-end MAS943SEQ ID NO: 7538 GGTGTGGTCGGCATATAGA OGL34 First PCR 5′-end MAS944SEQ ID NO: 7539 TTCGCACAAGCCATCC 3′-end MAS946 SEQ ID NO: 7540AACGACTTTTTGAATAGATGCT Nest PCR 5′-end MAS945 SEQ ID NO: 7541GCATTCCTTCTTGTCTCGT 3′-end MAS947 SEQ ID NO: 7542AACTTAGAGAAACTCATAACTCATC OGL35 First PCR 5′-end MAS948 SEQ ID NO: 7543TCATAGCTTCAAGGGATTCAC 3′-end MAS950 SEQ ID NO: 7544 GTTCATCAAAACACGCAAGANest PCR 5′-end MAS949 SEQ ID NO: 7545 CTCATGCCAACAAAAGCC 3′-end MAS951SEQ ID NO: 7546 GTAGTAACAAAAATGGATAACGCAG OGL36 First PCR 5′-end MAS936SEQ ID NO: 7547 TATCTGGCTTGAAGCTGAAT 3′-end MAS938 SEQ ID NO: 7548TTATTTCCTTCGTGGCTTCG Nest PCR 5′-end MAS937 SEQ ID NO: 7549CTCCACAATTTAGCATCCAAG 3′-end MAS939 SEQ ID NO: 7550 CGTCCATGTTTACTTGGCTAOGL37 First PCR 5′-en MAS952 SEQ ID NO: 7570 GTCATCATAATTGCTGTCCCA3′-end MAS954 SEQ ID NO: 7571 GGATGTGTGCCTGAGC Nest PCR 5′-end MAS953SEQ ID NO: 7572 CCTTCCTCGTGCCCTTA 3′-end MAS955 SEQ ID NO: 7573CCCCTAATCTCATCGCAAG OGL38 First PCR 5′-end MAS932 SEQ ID NO: 7551TCTGTTGATTCCTAATCGTAGC 3′-end MAS934 SEQ ID NO: 7552GTGATTGACATTTGTCTATAAGCA Nest PCR 5′-end MAS933 SEQ ID NO: 7553CCTCTTCACTGTGACTGAAC 3′-end MAS935 SEQ ID NO: 7554 TTTCGGCTTGACATTTCTTTCOGL39 First PCR 5′-end MAS956 SEQ ID NO: 7555 TGGCAAATCACACGGTC 3′-endMAS958 SEQ ID NO: 7556 ACTACCTTGCCCCTAAGATC Nest PCR 5′-end MAS957SEQ ID NO: 7557 TGCCACGACAAGAATTTCAT 3′-end MAS959 SEQ ID NO: 7558TGGTGTGATTCCAACGC

TABLE 10 List of all ″In″ primers for nested In-Out PCRanalysis of optimal genomic loci. First 3′-end GM_UnDo_3′FSEQ ID NO: 7559 PCR CAAATTCCCACTAAGCGCT Nest 3′-end GM_UnDo_3′_NSTSEQ ID NO: 7560 PCR TAAAGGTGAGCAGAGGCA

Deployment of the In-Out PCR assay in a protoplast targeting system wasparticularly challenging as large amounts of the plasmid DNA was usedfor transfection, and the large amount of DNA remains in the protoplasttargeting system and was subsequently extracted along with cellulargenomic DNA. The residual plasmid DNA may dilute the relativeconcentration of the genomic DNA and reduce the overall sensitivity ofdetection and can also be a significant cause of non-specific, aberrantPCR reactions. ZFN induced NHEJ-based donor insertion typically occursin either a forward or a reverse orientation. In-Out PCR analysis of DNAfor the forward orientation insertion often exhibited false positivebands, possibly due to shared regions of homology around the ZFN bindingsite in the target and donor that could result in priming and extensionof unintegrated donor DNA during the amplification process. Falsepositives were not seen in analyses that probed for reverse orientationinsertion products and therefore all targeted donor integration analysiswas carried out to interrogate reverse donor insertion in the RTA. Inorder to further increase specificity and reduce background, a nestedPCR strategy was also employed. The nested PCR strategy used a secondPCR amplification reaction that amplified a shorter region within thefirst amplification product of the first PCR reaction. Use of asymmetricamounts of “in” and “out” primers optimized the junctional PCR furtherfor rapid targeting analysis at selected genomic loci.

The In-Out PCR analysis results were visualized on an agarose gel. Forall soybean selected genomic loci of Table 12, “ZFN+donor treatments”produced a near expected sized band at the 5′ and 3′ ends. Control ZFNor donor alone treatments were negative in the PCR suggesting that themethod was specifically scoring for donor integration at the target siteof at least 32 of the optimal nongenic soybean genomic loci. Alltreatments were conducted in replicates of 3-6 and presence of theanticipated PCR product in multiple replicates (≥2 at both ends) wasused to confirm targeting. Donor insertion through NHEJ often produceslower intensity side products that were generated due to processing oflinearized ends at the target and/or donor ZFN sites. In addition, itwas observed that different ZFNs resulted in different levels ofefficiency for targeted integration, with some of the ZFNs producingconsistently high levels of donor integration, some ZFNs producing lessconsistent levels of donor integration, and other ZFNs resulting in nointegration. Overall, for each of the soybean selected genomic locitargets that were tested, targeted integration was demonstrated withinthe soybean representative genomic loci targets by one or more ZFNs,which confirms that each of these loci were targetable. Furthermore,each of the soybean selected genomic loci targets was suitable forprecision gene transformation. The validation of these soybean selectedgenomic loci targets were repeated multiple times with similar results,thus confirming the reproducibility of the validation process whichincludes plasmid design and construct, protoplast transformation, sampleprocessing, sample analysis.

CONCLUSION

The donor plasmid and one ZFN designed to specifically cleave soybeanselected genomic loci targets were transfected into soybean protoplastsand cells were harvested 24 hours later. Analysis of the genomic DNAisolated from control, ZFN treated and ZFN with donor treatedprotoplasts by in-out junctional PCR showed targeted insertion of theuniversal donor polynucleotide as a result of genomic DNA cleavage bythe ZFNs (Table 12). These studies show that the universal donorpolynucleotide system can be used to assess targeting at endogenoussites and for screening candidate ZFNs. Finally, the protoplast basedRapid Targeting Analysis and the novel universal donor polynucleotidesequence systems provide a rapid avenue for screening genomic targetsand ZFNs for precision genome engineering efforts in plants. The methodscan be extended to assess site specific cleavage and donor insertion atgenomic targets in any system of interest using any nuclease thatintroduces DNA double or single strand breaks.

Over 7,018 selected genomic loci were identified by various criteriadetailed above. The selected genomic loci were clustered using PrincipalComponent Analysis based on the ten parameters used for defining theselected genomic loci. A representative of the clusters in addition tosome other loci of interest were demonstrated to be targetable.

TABLE 12 Illustrates the results of the integration of a universal donorpolynucleotide sequence within the soybean selected genomic locitargets. As indicated by the * below, donor insertion within OGL37 wasonly confirmed by a PCR reaction of the 5′ and 3′ junction sequence.Target- able Cluster ZFN Donor Locus Name ID Location Assignment (pDAB#)(pDAB#) (Y/N) OGL01 soy_ogl_ Gm02: 1204801 . . . 1 124201 124280 Y 3081209237 OGL02 soy_ogl_ Gm02: 1164701 . . . 2 124221 124281 Y 307 1168400OGL03 soy_ogl_ Gm06: 43091928 . . . 3 125305 125332 Y 2063 43094600OGL04 soy_ogl_ Gm06: 11576991 . . . 4 125309 125330 Y 1906 11578665OGL05 soy_ogl_ Gm01: 51061272 . . . 5 124884 124290 Y 262 51062909 OGL06soy_ogl_ Gm16: 1298889 . . . 6 124234 123838 Y 5227 1300700 OGL07soy_ogl_ Gm12: 33610401 . . . 7 124257 123839 Y 4074 33611483 OGL08soy_ogl_ Gm10: 40763663 . . . 8 125316 125332 Y 3481 40764800 OGL09soy_ogl_ Gm03: 41506001 . . . 9 124265 123836 Y 1016 41507735 OGL10soy_ogl_ Gm03: 37707001 . . . 10 124273 123837 Y 937 37708600 OGL11soy_ogl_ Gm15: 42391349 . . . 11 124888 124290 Y 5109 42393400 OGL12soy_ogl_ Gm20: 36923690 . . . 12 124885 124291 Y 6801 36924900 OGL13soy_ogl_ Gm19: 49977101 . . . 13 124610 124294 Y 6636 49978357 OGL14soy_ogl_ Gm14: 5050547 . . . 14 124614 124845 Y 4665 5051556 OGL15soy_ogl_ Gm18: 55694401 . . . 15 124636 124293 Y 6189 55695900 OGL16soy_ogl_ Gm13: 23474923 . . . 16 124648 124292 Y 4222 23476100 OGL17soy_ogl_ Gm08: 7532001 . . . 17 121225 121277 Y 2543 7534800 OGL18soy_ogl_ Gm02: 1220301 . . . 18 121227 121278 Y 310 1222300 OGL19soy_ogl_ Gm07: 17194522 . . . 19 121233 121279 Y 2353 17196553 OGL20soy_ogl_ Gm06: 10540801 . . . 20 121235 121280 Y 1894 10542300 OGL22soy_ogl_ Gm09: 40167479 . . . 22 121238 121281 Y 3218 40168800 OGL24soy_ogl_ Gm10: 2950701 . . . 24 121234 121280 Y 3333 2951800 OGL25soy_ogl_ Gm08: 7765875 . . . 25 121249 121284 Y 2546 7767500 OGL28soy_ogl_ Gm18: 6057701 . . . 28 125324 125334 Y 5957 6059100 OGL30soy_ogl_ Gm11: 10146701 . . . 30 121265 121288 Y 3818 10148200 OGL31soy_ogl_ Gm17: 6541901 . . . 31 121271 121289 Y 5551 6543200 OGL33soy_OGL_ Gm02: 459032101 . . . 1 124666 123812 Y 684 45907300 OGL34soy_OGL_ Gm02: 458165943 . . . 9 124814 121937 Y 682 45818777 OGL35soy_OGL_ Gm02: 459105101 . . . 1 124690 123811 Y 685 45913200 OGL36soy_OGL_ Gm04: 45820631 . . . 2 124815 121937 Y 1423 45822916 OGL37*soy_OGL_ Gm04: 46095801 . . . 1 125338 124871 Y 1434 46097968 OGL38soy_OGL_ Gm14: 3816738 . . . 1 124816 121937 Y 4625 3820070 OGL39soy_OGL_ Gm19: 5311001 . . . 1 124842 124864 Y 6362 5315000

Lengthy table referenced here US10233465-20190319-T00001 Please refer tothe end of the specification for access instructions.

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US10233465B2). Anelectronic copy of the table will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

The invention claimed is:
 1. A soybean plant, soybean plant part, orsoybean plant cell comprising a recombinant nucleic acid molecule, saidrecombinant nucleic acid molecule comprising a nongenic soybean genomicsequence of at least 1 Kb determined to comprise the followingcharacteristics: a. the level of methylation of said nongenic sequenceis 1% or less; b. said nongenic sequence shares less than 40% sequenceidentity with any other sequence contained in the Glycine max genome; c.said nongenic sequence is located within a 40 Kb region of a known orpredicted expressive soybean coding sequence; and d. said nongenicsequence exhibits a recombination frequency within the soybean genome ofgreater than 0.01574 cM/Mb; wherein said nongenic sequence comprises a 1Kb sequence having at least 95% sequence identity with a sequenceselected from the group consisting of SEQ ID NO: 2639, SEQ ID NO: 3682,SEQ ID NO: 4050, SEQ ID NO: 4593, SEQ ID NO: 4622, SEQ ID NO: 4879, SEQID NO: 4932, SEQ ID NO: 5102, SEQ ID NO: 5122, SEQ ID NO: 5520, SEQ IDNO: 5698, SEQ ID NO: 6087, SEQ ID NO: 6515, SEQ ID NO: 6571, SEQ ID NO:6586, SEQ ID NO: 6775 and SEQ ID NO: 6935; and a DNA of interest,wherein the DNA of interest is inserted into said nongenic sequence. 2.The soybean plant, soybean plant part, or soybean plant cell of claim 1,wherein said nongenic sequence is 1 Kb to 5.73 Kb in length and containsno methylated cytosine residues.
 3. The soybean plant, soybean plantpart, or soybean plant cell of claim 1, wherein a 40 Kb region of nativesoybean genome comprising said nongenic sequence also comprises at leastone known or predicted soybean coding sequence, or a sequence comprisinga 2 Kb upstream and/or 1 Kb downstream sequence of a known soybean gene.4. The soybean plant, soybean plant part, or soybean plant cell of claim1, wherein said DNA of interest comprises a gene expression cassettecomprising an insecticidal resistance gene, herbicide tolerance gene,nitrogen use efficiency gene, water use efficiency gene, nutritionalquality gene, DNA binding gene, and selectable marker gene.
 5. Thesoybean plant, soybean plant part, or soybean plant cell of claim 1,wherein said DNA of interest comprises a gene expression cassettecomprising an insecticidal resistance gene or herbicide tolerance gene.6. A method of making a transgenic plant cell comprising a DNA ofinterest targeted to one nongenic soybean genomic sequence, the methodcomprising: a. selecting an optimal nongenic soybean genomic sequence ofat least 1 Kb, wherein said nongenic soybean genomic sequence comprisesthe following characteristics: ii). the level of methylation of saidnongenic sequence is 1% or less; ii). said nongenic sequence shares lessthan 40% sequence identity with any other sequence contained in theGlycine max genome; iii). said nongenic sequence is located within a 40Kb region of a known or predicted expressive soybean coding sequence;and iv). said nongenic sequence exhibits a recombination frequencywithin the soybean genome of greater than 0.01574 cM/Mb; further whereinsaid nongenic sequence comprises a 1 Kb sequence having at least 95%sequence identity with a sequence selected from the group consisting ofSEQ ID NO: 2639, SEQ ID NO: 3682, SEQ ID NO: 4050, SEQ ID NO: 4593, SEQID NO: 4622, SEQ ID NO: 4879, SEQ ID NO: 4932, SEQ ID NO: 5102, SEQ IDNO: 5122, SEQ ID NO: 5520, SEQ ID NO: 5698, SEQ ID NO: 6087, SEQ ID NO:6515, SEQ ID NO: 6571, SEQ ID NO: 6586, SEQ ID NO: 6775 and SEQ ID NO:6935; b. introducing a site specific nuclease into a plant cell, whereinthe site specific nuclease cleaves said nongenic soybean genomicsequence; c. introducing the DNA of interest into the plant cell; d.targeting the DNA of interest into said nongenic locus, wherein thecleavage of said nongenic sequence facilitates integration of thepolynucleotide sequence into said nongenic locus; and e. selectingtransgenic plant cells comprising the DNA of interest targeted to saidnongenic locus.
 7. The method of making a transgenic plant cell of claim6, wherein said site specific nuclease is selected from the groupconsisting of a zinc finger nuclease, a CRISPR nuclease, a TALEN, ahoming endonuclease or a meganuclease.
 8. The method of making atransgenic plant cell of claim 6, wherein said DNA of interest isintegrated within said nongenic locus via a homology directed repairintegration method.
 9. The method of making a transgenic plant cell ofclaim 6, wherein said DNA of interest is integrated within said nongeniclocus via a non-homologous end joining integration method.